What Is Endostatin?

Endostatin is currently the strongest and best experimental tumor angiogenesis inhibitor. It has received much attention in recent years. Currently, it has undergone phase and phase clinical trials in the United States and may become a new generation of antitumor. drug.

Endostatin

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Endostatin is currently the strongest and best experimental tumor angiogenesis inhibitor. It has received much attention in recent years. Currently, it has undergone phase and phase clinical trials in the United States and may become a new generation of antitumor. drug.

Chinese name

Endostatin

Foreign name

endostatin

Types of

Tumor angiogenesis inhibitor

biological functions

Inhibitory effect on vascular endothelial cells

Endostatin is an endogenous angiogenesis inhibitor isolated and purified from cultured mouse endothelial cell tumor (EOMA) supernatant in 1997 by O'Reilly et al. [1]. It is a 20 kd molecular weight protein. Amino acid sequence analysis showed that endostatin is the C-terminal part of 18 molecules of collagen with a total of 184 amino acids. Endostatin is a degradation product of collagen 18. The degradation process includes at least two steps of enzymatic hydrolysis. Elastase, cathepsin L, and matrix metalloproteinase may be involved in the enzymatic hydrolysis [2]. Further crystal structure analysis found that the surface of the endostatin structure has a basic region composed of 11 arginine residues, which is the heparin binding site, which explains the high affinity properties of endostatin for heparin. The region competes with angiogenic factors in combination with heparin, which plays a role in inhibiting angiogenesis. However, some studies have shown that the binding of endostatin to the vessel wall does not depend on the heparin binding site, and it has no competitive inhibitory effect with FGF-2 [3]. In addition, a zinc ion binding site consisting of three histidines at positions 1, 3, and 11 at the N-terminus and an aspartic acid residue at position 76 was found in the endostatin sequence. The N-terminus surrounds a dimer structure. The combination of endostatin with zinc ions was initially thought to be important for its anti-angiogenic activity, but later studies that removed zinc ion binding sites by genetic modification showed that endostatin inhibited endothelial cell migration and tumor growth without relying on zinc Ion binding site [4].

2.1 Inhibitory effect of endostatin on vascular endothelial cells Endostatin can specifically inhibit the proliferation of vascular endothelial cells under the induction of bFGF, inhibit the migration of endothelial cells, and induce apoptosis of endothelial cells, but for non-endothelial cells such as smooth muscle Cells, 3T3 fibroblasts, and Lewis lung cancer cells have no inhibitory effect [5]. Kim et al. [6] also proved that endostatin can inhibit the ability of human umbilical vein endothelial cells to penetrate the artificial basement membrane, and it has a dose-dependent relationship with the inhibitory effect.

2.2 Inhibitory effect of endostatin on angiogenesis At present, a variety of experiments can confirm that endostatin inhibits the growth of blood vessels, but has no effect on resting vascular tissues. O'Reilly et al. [1] showed that endostatin expressed by E. coli or baculovirus showed obvious inhibition on chicken embryo angiogenesis through chicken embryo chorioallantoic membrane (CAM) experiment, and no toxic reaction was seen. Bloch et al. [7] showed that endostatin does not affect wound healing, wound contraction, wound infection and wound epithelium regeneration in mice, but it can reduce the formation of granulation tissue. Yin et al. [8] injected recombinant lentivirus carrying endostatin gene into the joints of mice with early rheumatoid arthritis induced by TNF, and the results showed that endostatin can inhibit

The formation of intravascular angiogenesis and vascular crests slows down the progression of rheumatoid arthritis. This shows that endostatin can not only inhibit tumor angiogenesis, but also inhibit pathological vascular inflammation.

2.3 Inhibitory effect of endostatin on tumor growth and metastasis Many domestic and foreign scholars have demonstrated that endostatin can inhibit the growth and metastasis of a variety of solid tumors by using recombinant endostatin protein or by endostatin gene therapy experiments. effect. Bohen used mouse recombinant endostatin to almost completely inhibit the growth of primary tumors such as Lewis lung cancer, melanoma, cellulose, hemangioendothelioma, and renal cell carcinoma in mice.The tumor entered the dormant period after 6 cycles of treatment. The tumor had no recurrence, no metastases were found, and no drug resistance occurred. At the same time, at the cellular level, there are also experiments that show that endostatin can inhibit the migration of tumor cells in artificial basement membrane gels [6]. Perletti et al. Used a dimethylbenzoanthracene (DMBA) -induced rat breast cancer animal model and subcutaneously injected endostatin 20 mg / kg daily for 4 weeks to keep the tumor dormant for 4 weeks after drug withdrawal, indicating that endostatin It also has a significant inhibitory effect on the primary tumor.

3 Endostatin mechanism of action

At present, endostatin anti-angiogenesis therapy has achieved amazing results, but its mechanism of action has not been fully elucidated.

The possible mechanisms are:

By down-regulating the transcriptional activity of -catenin (-catenin), it inhibits the expression of cyclin D1 and causes G1 phase arrest of endothelial cells [9];

Down-regulate the expression of anti-apoptotic proteins Bcl-2 and Bcl-XL and induce apoptosis of endothelial cells [5];

Combined with matrix metalloproteinase 2 precursor protein (pro-MMP2) to form a stable complex, prevent the activation of pro-MMP2, and inhibit the catalytic activities of MMP2 and MMP1, thereby inhibiting the migration of endothelial cells [6];

Combined with tropomyosin, destroying the integrity of the microfilament structure, causing loss of cell motor function and inducing apoptosis [10];

inhibit c-myc expression and inhibit endothelial cell migration;

The heparin binding site binds to the SH2 region of the adaptor protein Shb receptor on the surface of endothelial cells, activates the tyrosine kinase signal transduction system, causes endothelial cell G1 phase arrest, and induces endothelial cell apoptosis;

Integrin 51 plays an important role in regulating bFGF-induced angiogenesis. Endostatin can directly bind to integrin 51, affect the adhesion of endothelial cells to the extracellular matrix, and inhibit the migration and growth of endothelial cells [11];

Inhibition of VEGF receptor KDR / Flk-1 tyrosine phosphorylation, thereby inhibiting the binding of VEGF to endothelial cells, and inhibiting the activity of extracellular signal-regulated kinase ERK induced by VEGF [12].

4 Endostatin antitumor angiogenesis treatment

4.1 Direct use of recombinant endostatin protein

At present, recombinant endostatin gene engineering expression systems mainly include E. coli expression system, yeast expression system, mammalian cell expression system and the like. The expression system of E. coli is high in expression, but most of the products exist in the form of inclusion bodies, which are insoluble and difficult to refold, which makes it inconvenient to use. Although the protein is soluble after refolding, a large amount of protein is lost during this process. The expression products of mammalian cells have high activity, but the expression level is low, which is difficult to purify. And yeast expression is high in yield, easy to purify, and high in activity.In particular, the Pichia pastoris expression system is widely used for the expression of endostatin. The endostatin currently used in phase I clinical trials in the United States is the expression product of Pichia . O'Reilly et al. [1] injected endothelin expressed in Escherichia coli into tumor-bearing mice, 2.5 mg / kg reduced Lewis lung cancer transplant tumor volume by 53%, and 10 mg / kg reduced tumor volume by 97%. When it increased to 20 mg / kg, the primary tumor almost completely atrophied, and endostatin at 0.3 mg / (kg · d) could completely inhibit the growth of Lewis lung cancer metastases.

4.2 Transduction of endostatin gene by vector

The endostatin gene is transduced through an appropriate vector to make it long-term and stable expression of endostatin with high biological activity in vivo, which effectively makes up for the lack of protein therapy. It has been proven that plasmids, liposomes, adenoviruses, retroviruses, adeno-associated viruses and lentiviruses are all effective vectors. Relative to non-viral vectors, higher endostatin plasma concentrations can be obtained after transfection with viral vectors. Feld2man et al. [13] injected the endostatin expressing adenovirus recombinant into MC38 adenocarcinoma mice via tail vein, and the mouse plasma endostatin concentration was as high as 1 770 ng / ml, making MC38 adenocarcinoma relatively less susceptible to adenovirus infection. The inhibition rate still reached 40%.

4. 3 other treatments

Joki et al. [14] transduced the endostatin gene into certain cells, and then encapsulated this endostatin-secreting cell into alginate to form microcapsules. After injected into the brain, cells can resist body rejection and tissues. The digestion of enzymes continuously secretes a certain effective concentration of endostatin and effectively inhibits the growth of gliomas. Similarly, the method of continuous administration by micropump can keep the concentration of endostatin in the body stable, and the antitumor effect is obviously stronger than intermittent administration. Domestic scholars Xu Genxing et al. [15] used Bifidobacterium adolescentes as a carrier to introduce the endostatin gene into Bifidobacterium and inject them into the tail vein of tumor mice. It was found that Bifidobacterium adolescentis exists only in solid tumors, and other normal tissues. Bifidobacterium adolescentis was not seen, and it also effectively inhibited tumor growth and angiogenesis. In addition, they also used human endostatin gene Bifidobacterium to make oral preparations. Clinical trials of multiple cases of advanced solid tumor volunteers have proven that they have a good effect on the treatment of liver cancer, gastric cancer, colon cancer, lung cancer and other solid tumors. Tumor effect.

5 Problems and Outlook

From the discovery of endostatin to the current phase II clinical trials, the progress achieved in just a few years has been very encouraging. Experiments show that endostatin specifically inhibits vascular endothelial cells. It has certain inhibitory effects on a variety of solid tumors that rely on angiogenesis, and has no drug resistance and obvious toxic and side effects, opening a new path for clinical treatment of tumors. As the mechanism of endostatin is fully elucidated, and how to produce and purify highly efficient and active recombinant endostatin protein on a large scale, how to extend the half-life of endostatin in the body, and how to choose a safe and efficient suitable carrier for gene therapy It is believed that how to properly select the indications and the treatment target will solve the problems of the series of problems. It is believed that endostatin will have greater application prospects for anti-angiogenic therapy of tumors.