Use of cd147 in the preparation of a medicament for treating prostate cancer

Abstract

The application discloses application of CD147 in preparation of a drug for treating prostate cancer. The application discloses and verifies, for the first time, a key role mechanism of CD147 in promoting glycolysis in prostate cancer cells through 'inhibiting a NOX4-ROS axis'. Specifically, by inhibiting the expression or function of CD147, the intracellular ROS level can be increased, glycolysis key enzyme activity and overall glycolysis metabolism are inhibited, and finally the effect of inhibiting the proliferation of prostate cancer cells is achieved. This provides a new role target and strategy for treating prostate cancer, especially castration-resistant prostate cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a new use of a transmembrane glycoprotein CD147, particularly its application in the preparation of drugs for regulating oxidative stress and glycolytic metabolism in prostate cancer cells, thereby inhibiting the proliferation of prostate cancer cells. Background Technology

[0002] Prostate cancer (PCa) is the most common malignant tumor in men worldwide. Androgen deprivation therapy (ADT) is the standard treatment for PCa. Within 2–3 years of receiving ADT, patients transform from androgen-dependent prostate cancer (ADPC) to castration-resistant prostate cancer (CRPC). At this point, traditional ADT treatment becomes ineffective, leading to tumor recurrence and metastasis, ultimately resulting in patient death, although the underlying mechanisms remain unclear. Increasing evidence suggests that oxidative stress and glycolysis are closely related to the development of PCa. Therefore, identifying the key molecules regulating oxidative stress and glycolysis in PCa has significant scientific and socioeconomic value.

[0003] Reactive oxygen species (ROS) play a crucial role in the life processes of the body. ROS primarily originate from the electron transport respiratory chain within mitochondria and enzymes such as nicotinamide purine dinucleotide phosphate oxidase (NOX) and xanthine oxidase. Mitochondria are the main site of ROS production in the body's cells, and the NOX family is the second largest source of ROS after mitochondria. The identified NOX family includes NOX1, NOX2, NOX3, NOX4, NOX5, and bifunctional oxidases, with NOX4 being the major subtype. NOX4 is essential for the survival and growth of PCa cells (especially androgen receptor-positive PCa cells). Compared to normal cells, tumor cells typically contain higher levels of ROS to maintain their proliferation and growth. However, if ROS accumulates continuously and exceeds the death threshold, it leads to apoptosis. Therefore, tumor cells have developed a robust endogenous antioxidant system to maintain ROS within a relatively stable and tolerable range, achieving adaptive survival under high oxidative stress and tolerance to the killing effects induced by chemotherapy drugs. Studies have found that ADT treatment can induce PCa cells to produce inhibitory levels of ROS, while also reducing the expression levels of antioxidant enzymes, leading to excessive ROS production and cell death. The emergence of the CRPC phenotype is also accompanied by an increase in the levels of cellular redox protective proteins. These results suggest that escaping the production of lethal doses of ROS during ADT treatment may lead to the development of CRPC. Further exploration of the molecular mechanisms regulating ROS production may provide new clues for CRPC treatment.

[0004] In recent years, research on energy metabolism in tumor cells has received increasing attention. Among these studies, glucose metabolism is considered to be the most specific alteration in tumor cell metabolism. Studies have shown that in the early stages of proliferative cysteine ​​(PCa), the zinc ion content in PCa cells decreases, weakening the inhibitory effect of zinc ions on aconitine synthase, leading to enhanced tricarboxylic acid (TCA) cycling and a decrease in the proportion of energy supplied by glycolysis. On the other hand, the expression level of the mitochondrial pyruvate transporter (MPC), a key protein controlling pyruvate influx into mitochondria, significantly increases in the early stages of PCa, closely related to the reopening of pyruvate influx and a relative increase in glucose oxidative phosphorylation. In the late stages of PCa, MPC expression is downregulated, resulting in obstruction of pyruvate influx into mitochondria and a renewed increase in glycolytic metabolism. Other studies have shown that in patients with a Gleason score greater than 7 or CRPC, the glucose metabolism pathway in tumor cells is mainly glycolysis. Therefore, in the early stages of PCa, tumor cell metabolism is mainly based on the TCA cycle, while as the tumor progresses and the malignancy of PCa cells increases, the dependence on the TCA cycle significantly decreases, shifting to primarily relying on aerobic glycolysis to provide energy for cell proliferation. The specific mechanisms require further investigation.

[0005] CD147 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Its expression is increased in various tumor cells, such as breast cancer and liver cancer, and its high expression is used as an indicator of poor clinical prognosis. Studies have shown that CD147 can promote aerobic glycolysis in hepatic stellate cells. Under hydrogen peroxide-induced oxidative stress, CD147 can improve the survival rate of malignant melanoma cells. However, whether CD147 promotes PCa progression through aerobic glycolysis and oxidative stress pathways, and the regulatory relationship between these pathways, remains poorly understood. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing a drug that can effectively treat prostate cancer, especially castration-resistant prostate cancer.

[0007] This invention, through in-depth research, reveals and verifies for the first time the key mechanism by which CD147 promotes glycolysis in prostate cancer cells by inhibiting the NOX4-ROS axis. Specifically, this invention provides the application of CD147 in the preparation of drugs for the treatment of prostate cancer.

[0008] Preferably, the prostate cancer is hormone-sensitive prostate cancer or hormone-resistant prostate cancer.

[0009] Preferably, knocking down CD147 promotes the production of reactive oxygen species (ROS) in prostate cancer cells.

[0010] Preferably, knockdown of CD147 has no significant effect on mitochondrial ROS production, but knockdown of CD147 can upregulate the expression and / or activity of NOX4 in prostate cancer cells, increase the level of reactive oxygen species in prostate cancer cells, thereby inhibiting glycolytic metabolism and / or proliferation of prostate cancer cells.

[0011] Preferably, the inhibition of glycolytic metabolism includes inhibiting the activity of pyruvate kinase (PK), thereby reducing lactate production and / or reducing ATP production.

[0012] Preferably, the NOX4 inhibitor partially reverses the increase in reactive oxygen species levels caused by CD147 knockdown.

[0013] Preferably, the NOX4-specific inhibitor is GKT137831.

[0014] Preferably, knocking down CD147 inhibits glycolysis by reducing pyruvate kinase activity by increasing reactive oxygen species levels in prostate cancer cells, thereby inhibiting the proliferation of prostate cancer cells.

[0015] Preferably, CD147 is knocked down by infecting a lentivirus that targets CD147 with shRNA.

[0016] Preferably, the shRNA sequence is as follows: 5'-GTCGTCAGAACACATCAACT-3'.

[0017] Preferably, treatment with the broad-spectrum ROS scavenger N-acetylcysteine ​​(NAC) can partially reverse the inhibition of glycolysis, reduced lactate / ATP production, and suppressed cell proliferation caused by CD147 knockdown.

[0018] CD147 primarily affects the activity of pyruvate kinase (PK) by regulating ROS levels, thereby influencing glycolytic flux. This invention, by inhibiting CD147 expression or function, can relieve its inhibition of NOX4, increase intracellular ROS levels, and thus inhibit the activity of key glycolytic enzymes and overall glycolytic metabolism, ultimately achieving the effect of inhibiting prostate cancer cell proliferation. This provides a new target and strategy for the treatment of prostate cancer, especially castration-resistant prostate cancer (CRPC). Attached Figure Description

[0019] Figure 1 The study showed the interference of CD147 expression in LNCaP and C4-2 cells. (A) Expression level of CD147 mRNA in LNCaP and C4-2 cell lines after transfection with knockdown lentivirus; (B) Changes in CD147 protein expression in LNCaP and C4-2 cell lines after transfection with knockdown lentivirus. ***P<0.001 compared to the control group.

[0020] Figure 2 The changes in ROS and CD147 expression in an AAPH-induced oxidative stress cell model are shown. (A) Detection of total ROS content in cells. (B) Detection of CD147 transcription level by RT-PCR. Compared with the control group, ***P<0.001; ****P<0.0001.

[0021] Figure 3 The study showed that CD147 knockdown promoted oxidative stress in LNCaP and C4-2 cells. Total ROS content (A), SOD activity (B), GSH-Px / GPX activity (C), and MDA level (D) were measured in LNCaP and C4-2 cells transfected with shNC (negative control) and shCD147, respectively. Compared with the shNC group, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0022] Figure 4The study showed that CD147 knockdown increases ROS production through NOX4-mediated reduction. (A) Mitochondrial ROS levels were measured in LNCaP and C4-2 cells transfected with shNC and shCD147, respectively. (B, C) NOX activity and NADP levels were measured in LNCaP and C4-2 cells transfected with shNC and shCD147, respectively. + / NADPH ratio. (D) Western blot detection of NOX4 antibody for immunoprobe labeling. Gray values ​​were normalized using β-actin as an internal control, and the fold change compared to the shNC group was marked below the corresponding band. (E) LNCaP and C4-2 cells transfected with shNC and shCD147 were treated with the NOX4 inhibitor GKT137831 for 24 hours, followed by detection of cellular ROS levels. Compared with the shNC group, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0023] Figure 5 The reactive oxygen species (ROS) scavenger NAC alleviated the glycolytic inhibition caused by CD147 knockdown in LNCaP and C4-2 cells. (A) LNCaP and C4-2 cells transfected with shNC and CD147-targeting shCD147 were treated with 5 mM NAC for 0 and 24 hours, and ROS levels were then measured. (BD) LNCaP (left) and C4-2 (right) cells transfected with shNC and shCD147 were treated with 5 mM NAC for 0 and 24 hours, and extracellular acidification rate (ECAR), lactate, and ATP levels were measured. (E) Pyruvate kinase (PK) activity was measured after NAC treatment. Hexokinase (HK) (F) and phosphofructokinase (PFK) (G) activities were measured after NAC treatment. (H) Several hours after NAC treatment, the cells were replaced with normal culture medium and cultured for 1, 3, and 5 days. Cell proliferation was measured using the CCK-8 assay. Compared with the NAC treatment group for 0 hours: **P<0.01, ***P<0.001, ****P<0.0001; compared with the shNC group: ****P<0.0001. Detailed Implementation

[0024] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0025] In the description of this invention, references to terms such as "some embodiments" and "examples" indicate that the specific methods or materials described in connection with that embodiment or example are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the specific methods and materials described may be combined in any suitable manner in one or more embodiments or examples.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0028] Example 1: Establishment of LNCaP and C4-2 cell lines with low CD147 expression To investigate the biological function of CD147 in PCa cells, CD147 was knocked down in LNCaP (hormone-sensitive prostate cancer) and C4-2 (hormone-resistant prostate cancer) cells by infecting them with shRNA lentiviruses that target CD147.

[0029] CD147 shRNA sequence: 5'-GTCGTCAGAACACATCAACT-3'.

[0030] LNCaP and C4-2 cells were seeded in culture plates. When the cell confluence reached 60%–70%, they were infected with CD147 interfering lentivirus constructed by Shanghai Jikai. Viral solution was added at the recommended MOI concentration (10) and supplemented with an infection enhancer. After 24 h of infection, the viral solution was discarded, and fresh complete culture medium was used for further culture for 72 h. Stable infected cell lines were obtained by puromycin selection. The inhibitory effect of CD147 expression was verified by qRT-PCR and Western blot, and subsequently used for related functional experiments.

[0031] Figure 1 This was observed to interfere with CD147 expression in LNCaP and C4-2 cells. Figure 1 As shown in A and B, qRT-PCR and Western blot results confirmed that after transfection with knockdown lentivirus, the expression levels of CD147 mRNA and CD147 protein in LNCaP and C4-2 cell lines decreased, indicating that CD147 was effectively knocked down in both LNCaP and C4-2 cells.

[0032] Example 2: Knocking down CD147 promotes NOX4 expression and increases ROS production 1. Knock down CD147 to promote ROS production LNCaP and C4-2 cells were treated with 4 mM 2,2'-azobis(2-amidinylpropane)dihydrochloride (AAPH) for 0, 48, and 72 hours, respectively, to induce ROS generation.

[0033] The expression changes of ROS and CD147 in AAPH-induced oxidative stress cell models were observed to further verify the effect of CD147 on ROS in LNCaP and C4-2 cells.

[0034] The results showed that after AAPH treatment, the total ROS levels of both cell types gradually increased as the treatment time increased from 48 hours to 72 hours. Figure 2 (A in the middle).

[0035] RT-PCR was used to detect the transcriptional level of CD147. The results showed that CD147 expression initially decreased and then increased, suggesting a negative correlation between CD147 and ROS in prostate cancer (PCa); excessive ROS promotes CD147 expression, thereby exerting a protective effect on cells. Figure 2 (B in the middle).

[0036] Figure 3 The results showed that knocking down CD147 increased the total ROS levels in both LNCaP and C4-2 cells. Figure 3 (A in the middle).

[0037] Total ROS content, SOD activity, GSH-Px / GPX activity and MDA level were measured in LNCaP and C4-2 cells transfected with shNC (negative control) and shCD147, respectively.

[0038] The results showed that knocking down CD147 reduced the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px / GPX), while increasing malondialdehyde (MDA) levels. Figure 3 (B, C, and D in the original text). MDA is a lipid peroxidation product and a marker of oxidative damage.

[0039] 2. Knocking down CD147 promotes ROS generation through the NOX4 pathway. Mitochondrial ROS changes in LNCaP and C4-2 cells of shNC and shCD147 were detected by flow cytometry (A). The results showed that ROS changes were not significant. NOX is another major source of intracellular ROS, which can catalyze the dehydrogenation of NADPH to NADP. +The generated electrons are then transferred to molecular oxygen, leading to the formation of superoxide anions. To investigate the mechanism by which knockdown of CD147 promotes ROS generation, NOx activity was measured.

[0040] NOX activity and NADP were measured in LNCaP and C4-2 cells transfected with shNC and shCD147, respectively. + / NADPH ratio. Figure 4 Results B and C showed that in LNCaP and C4-2 cells, knockdown of CD147 increased NOX activity and elevated NADP levels. + / NADPH ratio.

[0041] Western blot analysis was performed to detect NOX4 antibody-labeled immunosorbent assay (IRISA). Gray-scale values ​​were normalized using β-actin as an internal control, and the fold change compared to the shNC group was plotted below the corresponding bands. Results showed that knockdown of CD147 upregulated NOX4 expression levels in cells. Figure 4 (D in the middle).

[0042] LNCaP and C4-2 cells transfected with shNC and shCD147 were treated with the NOX4 inhibitor GKT137831 (Setanaxib, CAS No.: 1218942-37-0) for 24 hours, followed by detection of cellular ROS levels. The results showed that the NOX4 inhibitor GKT137831 could partially reverse the increase in ROS levels induced by CD147 knockdown. Figure 4 (E in the text).

[0043] Example 3: The reactive oxygen species scavenger NAC can alleviate glycolysis inhibition caused by CD147 knockdown. CD147 promotes glycolysis in PCa cells. Knockdown of CD147 induces glycolysis inhibition. Numerous studies have shown that abnormal production of reactive oxygen species (ROS) in various solid tumors can inhibit glycolysis by regulating key genes in the glycolytic pathway.

[0044] To investigate whether the inhibition of glycolysis induced by CD147 knockdown depends on elevated ROS levels, cells were treated with N-acetyl-L-cysteine ​​(NAC) to clear ROS, and then the glycolysis level was reassessed.

[0045] LNCaP and C4-2 cells transfected with shNC and shCD147-targeting NAC were treated for 0 and 24 hours, followed by ROS level detection. Results showed that NAC reduced ROS levels in LNCaP and C4-2 cells transfected with shNC and shCD147. Figure 5 (A in the middle).

[0046] LNCaP and C4-2 cells transfected with shNC and shCD147 were treated with 5 mM NAC for 0 and 24 hours. Extracellular acidification rate (ECAR), lactate, and ATP levels were then measured. Results showed that N-acetylcysteine ​​(NAC) could partially reverse the glycolytic inhibition caused by CD147 knockdown, as well as the reduced production of lactate and adenosine triphosphate (ATP). Figure 5 The data shows that knocking down CD147 inhibits glycolysis in prostate cancer (PCa) cells by increasing ROS levels.

[0047] The activity of pyruvate kinase (PK) was measured after NAC treatment. The activities of hexokinase (HK) and phosphofructokinase (PFK) were also measured after NAC treatment. The results showed that NAC treatment could partially alleviate the decrease in pyruvate kinase (PK) activity induced by CD147 knockdown, but had no significant alleviating effect on the decrease in hexokinase (HK) and phosphofructokinase (PFK) activity. Figure 5 (EG in the middle).

[0048] Several hours after NAC treatment, the cells were replaced with normal culture medium and cultured for 1, 3, and 5 days. Cell proliferation was then assessed using the CCK-8 assay. The results showed that NAC treatment could partially reverse the inhibitory effect of CD147 knockdown on the proliferation of both cell lines. Figure 5 (H in the text).

[0049] The above results indicate that inhibiting CD147 expression suppresses glycolysis by increasing ROS levels and reducing PK activity, thereby inhibiting the proliferation of prostate cancer cells.

Claims

1. Application of CD147 in the preparation of drugs for the treatment of prostate cancer.

2. The application according to claim 1, characterized in that, The prostate cancer mentioned is either hormone-sensitive prostate cancer or hormone-resistant prostate cancer.

3. The application according to claim 1 or 2, characterized in that, Knocking down CD147 promotes the production of reactive oxygen species in prostate cancer cells.

4. The application according to claim 1 or 2, characterized in that, Knockdown of CD147 had no significant effect on mitochondrial ROS production, but it could upregulate the expression and / or activity of NOX4 in prostate cancer cells, increase the level of reactive oxygen species in prostate cancer cells, and thus inhibit glycolytic metabolism and / or proliferation of prostate cancer cells.

5. The application according to claim 4, characterized in that, The inhibition of glycolytic metabolism includes inhibiting the activity of pyruvate kinase, thereby reducing lactate production and / or reducing ATP production.

6. The application according to claim 1 or 2, characterized in that, NOX4 inhibitors partially reverse the increase in reactive oxygen species levels caused by CD147 knockdown.

7. The application according to claim 6, characterized in that, The NOX4-specific inhibitor is GKT137831.

8. The application according to claim 1 or 2, characterized in that, Knocking down CD147 inhibits glycolysis by reducing pyruvate kinase activity and thus inhibiting the proliferation of prostate cancer cells by increasing reactive oxygen species levels in prostate cancer cells.

9. The application according to claim 1 or 2, characterized in that, CD147 was knocked down by infecting a lentivirus with shRNA that targets CD147.

10. The application according to claim 9, characterized in that, The sequence of the shRNA is as follows: 5'-GTCGTCAGAACACATCAACT-3'.

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