CN111281869B

CN111281869B - Application of melatonin in preparation of medicine for treating and assisting in treating osteosarcoma

Info

Publication number: CN111281869B
Application number: CN202010091027.1A
Authority: CN
Inventors: 张威林; 张治宇; 赵威
Original assignee: China Medical University
Current assignee: China Medical University
Priority date: 2020-02-13
Filing date: 2020-02-13
Publication date: 2022-08-05
Anticipated expiration: 2040-02-13
Also published as: CN111281869A

Abstract

The invention provides melatonin and application of a melatonin composition in preparation of a medicine for treating and adjunctively treating osteosarcoma. The invention discovers that the expression of a melatonin receptor MT1 in osteosarcoma cells is increased and the current phase that the CIC of the tumor cells is homogeneous is existed through experimental research, and the melatonin can be applied to inhibit the key pathway Rho/ROCK of the CIC through a cAMP/PKA signal pathway so as to inhibit the same CIC, intervene the mitochondrial physiology of the osteosarcoma cells and play a role in resisting the invasion and transfer of osteosarcoma. Finally provides the application of the melatonin and the better melatonin composition in the preparation of the medicine for treating and assisting in treating osteosarcoma, and provides a new treatment target point and direction for clinic.

Description

Application of melatonin in preparation of medicine for treating and assisting in treating osteosarcoma

Technical Field

The invention relates to the technical field of medicinal chemistry, in particular to application of melatonin in preparing a medicament for treating and adjunctively treating osteosarcoma.

Background

Melatonin (MT) is a neuroendocrine hormone secreted mainly from the pineal gland, and has various physiological functions such as circadian rhythm regulation, immunoregulation, antioxidation and anti-inflammation. In recent years, a great deal of literature indicates that MT has anti-tumor and chemopreventive effects, and research on the anti-tumor effect of MT has become a hot spot, wherein the research on tumors such as breast cancer, melanoma, colon cancer, prostate cancer and the like is included. Evidence suggests that various mechanisms are involved in the anti-tumor effects of MT, such as interfering with estrogen signaling and affecting telomerase activity. MT not only has single tumor inhibition effect, but also has the functions of synergism and toxicity reduction in tumor adjuvant therapy.

Osteosarcoma (OS) is the most common type of malignant bone tumor, which originates from abnormal transformation of mesenchymal cells, manifesting as osteoblast differentiation and generation of malignant bone-like substances. Mirabello et al showed, based on the national institute for tumor data, that OS most commonly occurs in the adolescent stage, with gender-related morbidity, with men having a higher incidence than women. Osteosarcoma occurs mainly at the metaphysis of long bones, and the disease has poor prognosis and strong invasiveness and brings great pain to patients. In this context, the use of a safe and effective anti-tumor agent in the treatment of osteosarcoma is becoming increasingly important.

Disclosure of Invention

In order to overcome the technical problems, the invention firstly provides the application of the melatonin in preparing the medicines for treating and assisting in treating the osteosarcoma.

The current clinical treatment scheme of OS is divided into three parts, namely preoperative neoadjuvant chemotherapy, intraoperative resection of primary and metastatic tumor areas and postoperative chemotherapy. OS cells are insensitive to radiation therapy. However, the existing chemotherapy drugs and antitumor drugs have the following defects: poor tissue specificity, inability to accurately locate OS tissues, and systemic toxic and side effects. Because the small-molecule chemotherapeutic drug has strong toxicity, the small-molecule chemotherapeutic drug has great influence on the health of human bodies while killing tumors. And the long-term use of the micromolecule chemotherapy drugs can cause the problems of drug effect reduction, multi-drug resistance and the like. At present, both chemotherapy schemes and surgical excision treatment schemes have high requirements on the basic constitution of patients, and the whole treatment process consumes the organisms of the patients greatly. The melatonin serving as a hormone naturally existing in a human body has the natural advantages of good biocompatibility and high safety, and the invention defines the exact treatment effect of the melatonin on osteosarcoma through in-vivo and in-vitro experiments, and can be used for preparing novel, effective and valuable medicine selection for treating OS. Meanwhile, the excellent safety of the melatonin is proved by the results of HE staining and pharmacokinetics of liver, lung and kidney tissues of experimental animals in a treatment group through in vivo experiments of mice.

In the research process, the invention discovers that melatonin is a treatment mode based on osteosarcoma tissue cell-in-cell (CIC) phenomenon, is different from the treatment ideas of the prior chemotherapy or antitumor drugs such as tumor cell poisoning or immunity activation and the like, is a method for inhibiting OS energy acquisition, is a treatment method with high specificity, has no side effect on normal tissue cells, and can replace the body with the treatment effect of effectively inhibiting tumor development at the minimum damage cost. The invention discovers that CIC phenomenon exists in osteosarcoma tissues of clinical patients, and determines that the type of CIC is phagocytosis type of allogenic cells in the pathological process of osteosarcoma. The correlation between melatonin and CIC has not been reported, but the relationship between Rho/ROCK and CIC structure has been found. Furthermore, Melatonin type 1 receptors (Melatonin Receptor 1, MT1) can activate the cAMP/PKA signaling pathway. There is a regulatory relationship between PKA and Rho. Therefore, melatonin activates a cAMP channel through a melatonin receptor, further activates PKA, and finally achieves the purpose of inhibiting Rho/ROCK, thereby interfering the formation of a CIC structure. The fact proves that the melatonin is also proved by experiments, so that the regulatory relation between the melatonin and the CIC is discovered for the first time. Then, the fact that CIC regulates osteosarcoma function through an energy metabolism pathway is verified by western blotting experiments, and mitochondrial function is consistent with CIC change in the process. Previous studies have shown that under extreme conditions, such as in a starved microenvironment, host cells can provide themselves with nutrients by engulfmg effector cells. Leibman et al observed degraded lymphocytes in host egg cells and suggested that degraded lymphocytes might provide nutrients to the host cells. Later Trowell et al proposed similar hypotheses that observed degraded lymphocytes in fibroblasts and thymic reticulocytes in vitro, Ioachim in various cells in vitro, and Fais et al found that metastatic melanoma cells could be maintained viable by the uptake of live T cells under nutrient-deficient conditions. In the experiments of the present invention, we demonstrated that the energy increase of OS cells after tumor phagocytosis can promote the proliferation and migration of OS, and that melatonin inhibits the energy metabolism of OS through the above-mentioned pathway to achieve the result of OS inhibition. In conclusion, we demonstrate that melatonin has a definite therapeutic effect on osteosarcoma and can be used for preparing a novel, effective and valuable drug option for treating OS.

Then, IN further experimental studies, we found that the efficacy of melatonin was enhanced by combining melatonin with a ROCK inhibitor, preferably ROCK-IN-1 (chemical formula: C), and confirmed this by experiments and the range of the amount of the ROCK inhibitor therebetween by experiments ₂₀ H ₁₈ FN ₃ O, CAS No.934387-35-6), the matching ratio of the melatonin and the melatonin is preferably 1: 3-3: 1, and most preferably 3: 1. Therefore, the invention further provides the application of the melatonin composition in preparing medicaments for treating and adjunctively treating osteosarcoma. Wherein the melatonin composition comprises melatonin and a ROCK inhibitor. The ROCK inhibitor is preferably ROCK-IN-1, and other known ROCK inhibitors that inhibit the activity of ROCK2 may be used. Wherein the weight ratio of the melatonin to the ROCK inhibitor is as follows: 3:1.

In clinical application, the melatonin composition can be prepared into pharmaceutical preparations, such as oral preparations or intravenous preparations, and the like, for more convenient administration.

The invention discovers that the expression of a melatonin receptor MT1 in osteosarcoma cells is increased and the current phase that the CIC of the tumor cells is homogeneous is existed through experimental research, and the melatonin can be applied to inhibit the key pathway Rho/ROCK of the CIC through a cAMP/PKA signal pathway so as to inhibit the same CIC, intervene the mitochondrial physiology of the osteosarcoma cells and play a role in resisting the invasion and transfer of osteosarcoma. Finally provides the application of the melatonin and the better melatonin composition in the preparation of the medicine for treating and assisting in treating osteosarcoma, and provides a new treatment target point and direction for clinic.

Drawings

Fig. 1 is an immunofluorescence result, wherein TT: tumor cell phagocytosis tumor cells, TL: tumor cells phagocytize leukocytes, MT: macrophages phagocytose tumor cells, and the pie chart shows that the phagocytosis types are different;

fig. 2 is the immunofluorescence results, wherein MT 1: melatonin receptor 1, MT 2: melatonin receptor 2;

FIG. 3 shows scratch test results;

FIG. 4 shows the results of a Transwell test;

FIG. 5 shows HE staining results;

FIG. 6 shows immunofluorescence results;

fig. 7 is a graph showing the variation of melatonin blood concentration with time, in which LM: low concentration melatonin, 10mg/kg, MM: medium melatonin, 100mg/kg, HM: high concentration melatonin, 1000 mg/kg;

FIG. 8 shows HE staining results;

FIG. 9 shows the weight change of mice in the control group with melatonin;

fig. 10 shows the experimental results of example 2.

Detailed Description

Embodiments of the present invention are described below with reference to the drawings. Elements and features depicted in one drawing or one embodiment of the invention may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that the figures and description omit representation and description of components or processes that are not relevant to the present invention, but known to those of ordinary skill in the art, for the sake of clarity.

The invention is further described below with reference to the figures and examples.

Example 1: experimental process and result of tumor inhibition effect of melatonin on osteosarcoma cells

We used E-cadherin to label tumor cells, CD45 to label leukocytes, CD68 to label macrophages, fluorescence stained tumor tissue samples from 30 cases of osteosarcoma patients and observed CIC types (tumor cells endocytosed tumor cells, tumor cells endocytosed leukocytes, macrophages endocytosed tumor cells) in the samples using confocal laser microscopy. We observed that the predominant CIC type in the tumor tissue of osteosarcoma patients was tumor cell-endocytosed tumor cells, 26 of 30 samples were observed. While leukocytes and macrophages were predominantly present in the tumor microenvironment, few co-localized with tumor cells, with only 3 and 5 observed cases, respectively. These results indicate that CIC is present in the tumor tissue of osteosarcoma patients and is of the type that tumor cells endocytose tumor cells. The results are shown in fig. 1, which is an immunofluorescence result, wherein TT: tumor cell phagocytosis tumor cells, TL: tumor cells phagocytize leukocytes, MT: macrophages engulf tumor cells, and the pie chart is the ratio of different phagocytic types.

We examined the expression of melatonin receptors MT1 and MT2 in 30 tumor tissues of osteosarcoma patients using immunofluorescence techniques, and we found that both MT1 and MT2 were expressed in tumor tissues of osteosarcoma patients, and these results provided the possibility of treating osteosarcoma with melatonin. The results are shown in FIG. 2. The immunofluorescence results of fig. 2 show melatonin receptor 1(MT1) and melatonin receptor 2(MT2) expression in tumor cells. Increasing melatonin MT1 receptor number in osteosarcoma cells

Next we verified the expression of melatonin receptors MT1 and MT2 in different osteosarcoma cell lines. We selected four osteosarcoma cell lines U2OS, MG63, hos, 143B, and used human osteoblast hfob1.19 as a control, and we found no statistical difference between MT1 and MT2 in hos cells compared to hfob 1.19. The expression of the MT1 receptor was significantly increased in U2OS, MG63 and 143B cells, while the expression of the MT2 protein was not statistically different from that of the control group. Among them, the difference in expression of MT1 protein was most significant in U2OS cells. Therefore, we speculate that the pathway of melatonin acting on osteosarcoma cells is probably through MT1, and we selected U2OS cells as the experimental object in the following experiments.

Second, the influence of melatonin on the invasion and transfer capacity of osteosarcoma cells CIC and osteosarcoma in vitro experiments

We used E-cadherin to mark tumor cells and observed using confocal laser microscopy and found that the number of CIC structures in MG63 cells was significantly reduced after melatonin treatment. Rho protein, a key protein for regulating CIC, positively regulates the formation of CIC structure. We performed a scratch test after treating MG63 cells with melatonin, melatonin in combination with a Rho agonist, and with a Rho inhibitor alone, and observed cell migration at time points 0, 24, 48, and 72 hours, respectively, as shown in fig. 3, and the results of the scratch test in fig. 3 show the effect of melatonin and ROCK agonists and inhibitors on tumor cell invasion and migration at 12, 24, and 48 hours. We found that the group of ROCK inhibitors and melatonin groups had longer scratch distances, while the group of melatonin in combination with the ROCK agonist and the control group had smaller scratch distances. And the cell scratches of the melatonin group and the Rho inhibitor group hardly shrink after 24 hours, which indicates that the melatonin can play an inhibitory effect on the migration of osteosarcoma cells for a longer time. The Transwell experiment is carried out by adopting the same group, the result is shown in figure 4, and the Transwell experiment result of figure 4 shows that the influence of melatonin and ROCK agonists and inhibitors on the invasion and migration capacity of tumor cells, so that the cell migration quantity of the melatonin group and the Rho inhibitor group is obviously less than that of the melatonin combined Rho pathway agonist group and the control group. These results indicate that melatonin can inhibit CIC structure formation and possibly further inhibit osteosarcoma invasion metastatic ability through its inhibitory effect on CIC.

Third, the inhibitory action of melatonin on osteosarcoma in vivo experiments of mice

We used U2OS cells to make a mouse subcutaneous tumor model and found that melatonin effectively inhibited the growth of osteosarcoma in mice. HE staining was performed on mouse osteosarcoma tissues, as shown in FIG. 5, and the HE staining results of FIG. 5 show CIC of tumor cells in melatonin and control groups. The phenomenon of homogeneous CIC of osteosarcoma cells is found in tumors of a control group, and the quantity of CIC structures in the control group is obviously higher than that in a melatonin treatment group. The number of CIC phenomena in the specimens of the treatment group and the control group in the section was observed under a confocal laser microscope, the results are shown in FIG. 6, and the immunofluorescence results in FIG. 6 show the CIC conditions of the melatonin and the tumor cells in the control group. The frequency of the same species CIC phenomenon in the tissues of the control group is obviously higher than that in the treated group. From the above results, we found the inhibitory effect of melatonin on the growth of CIC and osteosarcoma in vivo experiments.

Fourth, the mechanism of melatonin for regulating CIC

We treated U2OS cells with melatonin and melatonin receptor agonists and inhibitors and further tested Sac, cAMP, PKA and Rho/ROCK levels. We found that melatonin and melatonin receptor agonists can up-regulate cAMP and PKA phosphorylation levels and inhibit the Rho/ROCK signaling pathway, exerting inhibitory effects on CIC. The use of melatonin receptor inhibitors based on melatonin may reverse the above results. Based on this, we found that PKA inhibitors can attenuate the inhibitory effect of melatonin on the Rho/ROCK signaling pathway. The results show that melatonin can play an inhibition role on a key signal pathway Rho/ROCK of CIC by activating a cAMP/PKA signal pathway, so that CIC is effectively inhibited.

Fifth, the Effect of CIC on the mitochondrial physiology of osteosarcoma cells

By transfecting CIC key protein Rho with lentivirus, we observe that the application of melatonin or silencing Rho can reduce the number of mitochondria in osteosarcoma cells, and can reduce the expression of mitochondrial biogenesis related proteins PGC1 alpha, NRF1 and TFAM and mitochondrial markers TOMM20, COX4l1 and ATP Syn beta, and the stimulation of Rho on the basis of the application of melatonin can reverse the effect of the melatonin. The results show that melatonin can inhibit mitochondrial biogenesis and mitochondrial function in osteosarcoma cells through a Rho/ROCK-mediated CIC pathway.

Sixth, melatonin pharmacokinetics and in vivo safety

Pharmacokinetic studies of melatonin in rats: melatonin is injected into the tail vein of a female rat according to mg/kg of body weight, and the concentration change condition of the melatonin in venous blood is observed by adopting a mode of eyeground vein blood collection. The drug concentrations at different time points (5min, 10min, 30min, 1h, 3h, 6h, 12h, 24h) are shown in fig. 7. Fig. 7 shows the trend of melatonin blood concentration over time, in which: LM (low melatonin concentration, 10mg/kg), MM (medium melatonin concentration, 100mg/kg), HM (high melatonin concentration, 1000mg/kg), as seen from the change in melatonin concentration in venous blood, the melatonin reached a peak in venous blood of rats after administration for 30min, and thereafter, continued to decrease only slightly up to 6 hours, with the drug concentration gradually decreasing from 6 hours later, and the melatonin content almost disappeared after 24 hours.

In vivo safety of melatonin: the safety of melatonin in mice was studied, and the important organs of the mice, such as heart, liver, spleen, lung and kidney, were collected once every 2 days after 30 days by injecting melatonin into the tail vein of the mice, and then subjected to HE staining of tissue sections, and the organ sections were observed under a mirror, as shown in FIG. 8, wherein the HE staining result of FIG. 8 shows that no organ damage is caused by the application of melatonin and the heart, liver, spleen, lung and kidney of the control mice. No organ damage was observed in melatonin-injected mice. The weight of the mice is measured as shown in fig. 9, and fig. 9 shows that the weight of the mice in the melatonin and control group is changed, and all the weights of the mice are stable without special decline and rise.

Example 2: experimental process and result of tumor inhibition effect of melatonin composition on osteosarcoma cells

Example 1 the results of the experiment in figure 3 demonstrate that the combined use of inhibitors can reduce the metastatic capacity of osteosarcoma cells. Therefore, the invention further performs experiments on the basis of example 1, and proves that the drug effect of the melatonin can be enhanced by combining the melatonin with a ROCK inhibitor, wherein the ROCK inhibitor is preferably ROCK-IN-1 (chemical formula: C) ₂₀ H ₁₈ FN ₃ O, CAS No.934387-35-6), the matching ratio of the melatonin and the melatonin is preferably 1: 3-3: 1, and most preferably 3: 1. Other known ROCK inhibitors that inhibit ROCK2 activity may also be used.

Melatonin and a ROCK inhibitor (ROCK-IN-1) compound preparation are applied to the medicine according to the weight ratio of 10mg/kg body weight (the weight ratio of the melatonin to the ROCK inhibitor is 1:1, 3:1 and 1:3 respectively). And combines the effects of intravenous and oral administration on the size of mouse osteosarcoma tumors. Tumor tissue is shown as tumor specimens after one week of once-daily continuous dosing. FIG. 10 shows the results of the experiment in which the horizontal axes of the bar graphs represent 1-6 in the left graph, respectively, and the vertical axes represent the ratio of tumor size to 1:1 intravenously administered group, and we found that when melatonin was intravenously administered in a ratio of melatonin to ROCK inhibitor 3:1, the tumor volume was minimal, and the compound preparation was most effective against osteosarcoma.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, devices, means, methods, or steps.

Claims

1. Use of a melatonin composition for the manufacture of a medicament for the treatment and co-treatment of osteosarcoma, wherein the melatonin composition comprises melatonin and a ROCK inhibitor, wherein the ROCK inhibitor is ROCK-IN-1; wherein the weight ratio of the melatonin to the ROCK inhibitor is as follows: 3:1.

2. A pharmaceutical preparation for the treatment and co-treatment of osteosarcoma, comprising melatonin and a ROCK inhibitor, wherein the ROCK inhibitor is ROCK-IN-1; wherein the weight ratio of the melatonin to the ROCK inhibitor is as follows: 3:1.

3. The pharmaceutical formulation of claim 2, wherein the formulation is an oral formulation.

4. The pharmaceutical formulation of claim 2, wherein the formulation is an intravenous formulation.