Application of MK2 in muscle atrophy caused by cancer cachexia

Abstract

The present invention belongs to the field of clinical medicine, specifically to the application of MK2 in muscle atrophy caused by cancer cachexia. The present invention discloses the role of the MK2 target in muscle atrophy caused by cancer cachexia, opens up new ideas for the study of the pathogenesis of cachexia, and also provides a potential new target for the treatment of cachexia.

Description

Technical Field

[0001] The present invention belongs to the field of clinical medicine, and particularly relates to the application of MK2 in muscle atrophy caused by cancer cachexia. Background Art

[0002] Improving survival and quality of life are intrinsic goals of successful cancer treatment and a desired translational outcome of basic cancer research. Cachexia has a clear adverse impact on these therapeutic goals. Cachexia, also known as caching, is a multifactorial wasting syndrome characterized by extreme weight loss, anemia, weakness, extreme pain, and systemic exhaustion. Cancer cachexia is characterized by persistent skeletal muscle loss (with or without fat loss), which cannot be fully reversed by traditional nutritional support and leads to progressive functional impairment. Cancer cachexia is a common clinical condition in the late stages of cancer, affecting approximately 60% of cancer patients and rising to 80% in the late stages. Cachexia severely reduces patients' quality of life, compromises cancer treatment outcomes, increases the incidence of cancer complications, and is a significant cause of death in patients with advanced cancer. Despite extensive animal, preclinical, and clinical research on cachexia over the past decade, there are still no effective drugs to treat cancer patients with cachexia, primarily due to the lack of clarity regarding the pathogenesis of cancer cachexia.

[0003] Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2 or MK2) is associated with many proteins. For example, MK2 is a downstream substrate of p38 MAPK and is responsible for multiple signaling cascades in response to a large number of extracellular stimuli, including apoptosis, cell division and differentiation, cell activity and inflammation.

[0004] In recent years, the molecular mechanisms of muscle wasting in cancer cachexia have been extensively studied in experimental animals, including those related to muscle protein degradation. Within the muscle protein degradation pathway, muscle wasting induced by various catabolic stimuli is partly due to overactivation of the ubiquitin-proteasome pathway (UPP) and the autophagy-lysosome pathway (ALP), which accelerates muscle protein degradation. However, unlike muscle wasting induced by disuse, fasting, and denervation, cancer cachexia is also characterized by severe systemic inflammation, which is considered a major factor in cancer-associated muscle wasting. Preclinical studies have identified multiple inflammatory cytokines found in the cancer milieu, including TNF, IL-6, IL-1, and Activin A / B, which stimulate muscle protein degradation through UPP and ALP. The underlying mechanism is believed to involve activation of signaling molecules that promote muscle catabolism, such as NF-κB, p38 mitogen-activated protein kinase (MAPK), STAT3, or SMAD2. However, cytokines operate in networks, and intervening in individual cytokines has not produced satisfactory results in clinical trials. Identifying intracellular signaling pathways shared by multiple cytokines that are critical for cancer-induced muscle atrophy may be a better therapeutic strategy.

[0005] The prior art does not disclose the role of MK2 in the development and progression of cancer cachexia. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of MK2 in cancer cachexia and muscle atrophy.

[0007] The present invention discovered that p38 MAPK activates the transcription factor C / EBPβ through two complex steps. First, p38 MAPK activates the acetyltransferase activity of p300 by phosphorylating its Ser12 residue. Activated p300 then acetylates C / EBPβ at Lys39 on C / EBPβ, thereby activating it. Second, p38 MAPK directly phosphorylates C / EBPβ at Thr188, thereby activating its DNA binding activity. Activated C / EBPβ binds to the promoters of key genes, including Atrogin1 and UBR2, key E3 ligases in the UPP pathway, and LC3b and Gabarapl1 in the ALP protein degradation pathway, upregulating the expression of these target genes. This leads to the loss of myosin heavy chain (MHC) and p62, specific substrates for these two protein degradation pathways. Therefore, the present invention suggests that p38 MAPK, p300, and C / EBPβ, respectively, are required for muscle atrophy in tumor-bearing mice, and that these signaling molecules are potential therapeutic targets for cancer cachexia.

[0008] In addition, when studying the upstream sources of p38βMAPK activation in cancer cachexia, the present invention found that in cachectic mice and human cancer cells, including lung cancer, pancreatic cancer, colon cancer, and gastric cancer, high levels of Hsp70 / 90 are continuously released via exosomes, inducing muscle atrophy by directly activating the TLR4 receptor on muscle cells. In addition, Hsp70 and Hsp90, through systemic activation of TLR4, lead to increased circulating inflammatory cytokines such as TNFα and IL-6. Therefore, TLR4 is required for cancer-induced p38MAPK activation and muscle atrophy. As a common effector of TLR4 and multiple cytokines (including TNFα, IL-6, IL-1, and Activin A), p38βMAPK is an important intracellular mediator of cancer-induced muscle atrophy.

[0009] Therefore, based on the above research findings, the present invention proposes the use of MK2 in cancer cachexia and muscle atrophy.

[0010] Specifically, MK2 is used as a target in the preparation of drugs for improving muscle atrophy caused by cancer cachexia.

[0011] Preferably, the drug is an oral preparation.

[0012] Preferably, the drug is an inhalation preparation.

[0013] Preferably, the drug is an injectable preparation.

[0014] Furthermore, the present invention also provides the use of an MK2 inhibitor in the preparation of a drug for treating cancer cachexia and muscle atrophy.

[0015] Wherein, the MK2 inhibitor contains siRNA or shRNA that specifically targets MK2.

[0016] Use of an MK2 inhibitor in the preparation of a drug inhibitor, wherein the drug inhibitor is any one or more of the following:

[0017] (1) Inhibit the increase in C / EBPβ phosphorylation caused by CCM;

[0018] (2) inhibition of CCM caused the increase of UBR2 and Atrogin1;

[0019] (3) reduce the degradation of MHC;

[0020] (4) inhibiting tumor-induced phosphorylation of MK2 and C / EBPβ;

[0021] (5) Inhibit muscle atrophy caused by tumor cachexia;

[0022] (6) Inhibit muscle protein degradation in tumor-bearing mice.

[0023] Beneficial effects of the present invention:

[0024] The present invention discloses the role of the MK2 target in muscle atrophy caused by cancer cachexia, opens up new ideas for the study of the pathogenesis of cachexia, and also provides a potential new target for the treatment of cachexia. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This study demonstrates that MK2 regulates the activation of the protein degradation pathway in muscle cells. MK2 was knocked down by MK2-specific siRNA, and C2C12 myotubes were treated with CCM for 1, 8, and 72 hours. C2C12 cell lysates were collected and immunoblotted to detect MK2 and C / EBPβ phosphorylation levels (A), UBR2 and Atrogin1 levels (B), and changes in MHC protein (C).

[0027] Figure 2This study showed that MK2 inhibitors can inhibit the activation of protein degradation pathways in muscle cells. C2C12 myotubes were treated with MK2 inhibitor (PF-364402) for 1, 8, and 72 hours. Cell lysates were collected and analyzed by Western blotting to detect changes in C / EBPβ phosphorylation, UBR2, Atrogin1, and MHC protein.

[0028] Figure 3 Inhibition of MK2 has been shown to suppress activation of the skeletal muscle protein degradation pathway and muscle atrophy in tumor-bearing mice. C26 tumor-bearing mice were treated with the MK2 inhibitor (PF-364402, 10 mg / kg / day). Serum and skeletal muscle tissue were collected on day 21 and analyzed for MK2 phosphorylation, C / EBP phosphorylation, and protein levels of UBR2, Atrogin1, and MHC. DETAILED DESCRIPTION

[0029] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0030] Example 1 In vitro experiment

[0031] (1) C2C12 cell culture:

[0032] C2C12 cells were cultured at 37° C. and 5% CO 2 in DMEM containing 10% (v / v) fetal bovine serum (FBS) and 0.1% (w / v) gentamicin / ampicillin.

[0033] When the cell density reached 90%, the cell culture medium was replaced with differentiation medium (DMEM containing 4% horse serum), and culture was continued at 37° C. for 4 days to allow the C2C12 cells to differentiate into myotubes.

[0034] (2) Preparation of tumor cell conditioned medium:

[0035] Mouse colon cancer cells C26 were cultured at 37°C and 5% CO2 in RPMI-1640 containing 10% (v / v) fetal bovine serum (FBS) and 0.1% (w / v) gentamicin / ampicillin.

[0036] When the cell density reached 40%, the cell culture medium was replaced with fresh one and cultured at 37° C. for 48 hours. The cell culture medium was collected and centrifuged at 1000 rpm for 10 minutes. The supernatant was the C26 tumor cell conditioned medium (CCM).

[0037] (3) Treatment of C2C12 myotubes with tumor cell conditioned medium:

[0038] CCM and C2C12 differentiation medium were mixed at a ratio of 1:3 and treated with C2C12 myotubes for 1 hour and 8 hours. C2C12 cell lysates were collected and immunoblotting was used to detect MK2 and its phosphorylation levels, C / EBPβ phosphorylation, UBR2, and Atrogin1 protein changes.

[0039] The results are as follows Figure 1 As shown in A, after CCM treatment of C2C12 myotubes for 1 hour, the phosphorylation levels of MK2 and C / EBPβ increased. Knockdown of MK2 using MK2-specific siRNA inhibited the increase in C / EBPβ phosphorylation induced by CCM.

[0040] (4) Small interfering RNA (siRNA) transfection:

[0041] C2C12 cells were seeded in 6-well plates 24 hours before transfection and transfection was performed when the cells reached 50% density.

[0042] JetPRIME transfection reagent (Polyplus-transfection Inc., Illkirch, France) was used to transfect C2C12 cells with pre-designed MK2-specific siRNA (purchased from Shanghai Gene Pharmaceutical Technology Co., Ltd.) according to the manufacturer's instructions (100 nM siRNA added to 4 μl jetPRIME). Fresh culture medium was replaced overnight after transfection.

[0043] When the cell density reached 90%, the cell culture medium was replaced with differentiation medium (DMEM containing 4% horse serum) and cultured at 37°C for 4 days to allow the C2C12 cells to differentiate into myotubes. The C2C12 myotubes were treated with CCM for 1 hour, 8 hours, and 72 hours, and the C2C12 cell lysates were collected.

[0044] Western-blot was used to detect the levels of MK2 and its phosphorylation, C / EBPβ phosphorylation, UBR2, Atrogin1, and MHC protein changes.

[0045] like Figure 1As shown in B, after C2C12 myotubes were treated with CCM for 8 hours, UBR2 and Atrogin1 increased, and MK2 siRNA could inhibit the increase of UBR2 and Atrogin1 induced by CCM.

[0046] like Figure 1 As shown in C, after C2C12 myotubes were treated with CCM for 72 h, MK2 siRNA could inhibit the degradation of myosin heavy chain (MHC) induced by CCM.

[0047] (5) Treatment of C2C12 cells with MK2 inhibitors:

[0048] C2C12 myotubes were treated with MK2 inhibitor (PF-364402) for 1 hour and 8 hours, and C2C12 cell lysates were collected. Changes in C / EBPβ phosphorylation, UBR2, Atrogin1, and MHC protein were detected by Western blotting.

[0049] The results are as follows Figure 2 As shown in the results, after C2C12 myotubes were treated with CCM, MK2 inhibitor could inhibit the CCM-induced increase in C / EBPβ phosphorylation, inhibit the increase in UBR2 and Atrogin1, and alleviate the degradation of MHC.

[0050] Example 2 Animal Experiment

[0051] (1) Effect of MK2 inhibition on muscle atrophy in C26 tumor-bearing mice

[0052] Eight-week-old male BALB / c mice were injected subcutaneously with 1x10 6 One week later, C26 cells were treated with an MK2 inhibitor (PF-364402, 10 mg / kg / day) using a subcutaneously embedded drug sustained-release pump.

[0053] On the 21st day, the serum and skeletal muscle tissue of mice were collected, and the protein levels of MK2 phosphorylation, C / EBP phosphorylation, UBR2, Atrogin1, and MHC were detected. Correlation analysis was performed with tumor size, mouse weight changes, and muscle strength changes to determine the activation changes of various biological markers and signaling pathways in protein degradation pathways during tumor development and to observe whether MK2 inhibitors can slow or inhibit the progression of cancer cachexia.

[0054] (2) Histochemical staining to determine changes in muscle fibers

[0055] The muscle tissue of mice was fixed with neutral buffered formalin, embedded in paraffin, and then sectioned and stained with HE to observe the changes in muscle fiber thickness.

[0056] (3) Western blot

[0057] Approximately 10 mg of muscle tissue was extracted and homogenized for protein quantification using the BCA assay. SDS-PAGE gels were prepared based on the molecular weight of the target protein, and sample loading, electrophoresis, and membrane transfer were performed according to the experimental design. After blocking with skim milk, the corresponding primary and secondary antibodies were incubated. Chemiluminescence imaging and fixation were performed, and relative quantitative analysis of band density and range was performed using an internal control. Changes in MK2 phosphorylation, C / EBPβ phosphorylation, UBR2, and Atrogin1 proteins were detected.

[0058] (4) Real-time PCR

[0059] Tissue was lysed using Trizol solution (approximately 5 mg of tissue plus 1 ml of Trizol), and total RNA was extracted and reverse transcribed into cDNA using RT-PCR. qPCR primers for specific genes were designed and amplified on a fluorescence quantitative PCR instrument to detect mRNA expression levels. The primers used were:

[0060]

[0061] The results are as follows Figure 3 As shown, in the skeletal muscle tissue of tumor-bearing mice, phosphorylation levels of MK2 and C / EBPβ were elevated, as were UBR2 and Atrogin1. This was accompanied by a decrease in MHC, reduced muscle weight, and thinning of muscle fibers. Application of an MK2 inhibitor suppressed tumor-induced phosphorylation of MK2 and C / EBPβ, and the increase in UBR2 and Atrogin1, significantly improving muscle protein degradation and suppressing muscle atrophy associated with tumor cachexia in tumor-bearing mice.

[0062] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Use of MK2 as a target in the preparation of a drug for improving muscle atrophy caused by cancer cachexia, characterized in that: The drug is any one or more of the following: (1) Reduce the degradation of MHC; (2) inhibiting tumor-induced phosphorylation of MK2 and C / EBPβ; (3) Inhibit tumor cachexia and muscle atrophy.

2. Use of an MK2 inhibitor in the preparation of a drug for treating cancer cachexia and muscle atrophy, characterized in that: The drug is any one or more of the following: (1) Reduce the degradation of MHC; (2) inhibiting tumor-induced phosphorylation of MK2 and C / EBPβ; (3) Inhibit tumor cachexia and muscle atrophy.

3. The use according to claim 2, characterized in that The MK2 inhibitor contains siRNA or shRNA that specifically targets MK2.

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