Recombinant Human Endostatin

Recombinant human endostatin (rhEs) offers several compelling advantages for research applications, particularly in cancer biology and angiogenesis studies.

Anti-Angiogenic Efficacy

Recombinant human endostatin functions as a potent endogenous angiogenesis inhibitor with well-characterized mechanisms of action. It suppresses tumor formation through multiple pathways, including the inhibition of blood vessel growth. The protein works by preventing vascular endothelial growth factor (VEGF) from binding to endothelial cells through vascular endothelial growth factor receptors (VEGFR), effectively blocking VEGF signaling and inhibiting VEGF-mediated endothelial cell migration. Additionally, it directly downregulates the mRNA and protein expressions of VEGF, further suppressing angiogenic processes.

Demonstrated Biological Activity

In vitro studies have shown that rhEs significantly inhibits human umbilical vein endothelial cell (HUVEC) proliferation at concentrations of 2 µg/mL and above. The protein induces endothelial cell apoptosis, stops the cell cycle, and suppresses endothelial cell proliferation and migration through complex signaling systems. These effects make it valuable for investigating endothelial cell behavior and vascular development in controlled experimental settings.

Combination Therapy Potential

A major advantage of rhEs is its ability to normalize tumor vasculature, which creates a therapeutic window for enhanced chemotherapy efficacy. When combined with chemotherapeutic agents like paclitaxel, rhEs transiently improves anti-tumor efficacy by reducing tumor microvascular density, decreasing hypoxic fractions, and increasing pericyte coverage around endothelial cells. This vasculature normalization also improves tumor oxygenation, providing rationale for combined therapy approaches with chemoradiotherapy.

Safety Profile

Recombinant endostatin suppresses different tumor types with low toxicity and minimal drug resistance during long-term application, making it suitable for extended experimental protocols without concerns about cumulative toxicity or rapid resistance development.

Research Flexibility

The protein is available in multiple formulations—with or without carrier proteins like bovine serum albumin—allowing researchers to select the appropriate variant for their specific applications, whether for cell culture, tissue culture, or use as an ELISA standard.

You can use recombinant human Endostatin as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is well-characterized, matches the endogenous protein in immunoreactivity, and is compatible with your assay system.

Key considerations and supporting details:

• Parallelism and Linearity: ELISA kits designed for human Endostatin typically use E. coli-expressed recombinant human Endostatin as the calibrator or standard. Validation data from commercial kits show that standard curves generated with recombinant Endostatin are parallel to those obtained with natural (endogenous) Endostatin, indicating that the recombinant protein can serve as a reliable standard for quantification. This parallelism is essential for accurate quantification, as it demonstrates that the assay detects both forms equivalently.

• Calibration and Specificity: The calibration of these assays is often performed using highly purified recombinant human Endostatin, and the assays are validated to recognize both endogenous and recombinant forms. This ensures that quantification using recombinant standards reflects the concentration of natural Endostatin in biological samples.

• Validation Requirements: For best practice, ensure that:

  • The recombinant Endostatin is of high purity and its concentration is accurately determined.
  • The recombinant protein is free from tags or modifications that could affect antibody recognition, unless your assay has been validated for the specific recombinant construct you are using.
  • The assay demonstrates acceptable dilution linearity and recovery when recombinant Endostatin is spiked into your sample matrix.

• Assay Compatibility: If you are using a commercial ELISA kit, check the kit documentation to confirm that it is validated for both recombinant and endogenous Endostatin. Most reputable kits provide this information and often include data on recovery, linearity, and parallelism for recombinant standards.

• Research Use Only: Note that both recombinant Endostatin and most ELISA kits are for research use only and not for diagnostic procedures.

Summary Table: Recombinant Endostatin as ELISA Standard

In summary: You can use recombinant human Endostatin as a standard for ELISA quantification, provided it is well-characterized and matches the immunoreactivity of the endogenous protein in your assay system. Always verify compatibility and validation data for your specific assay.

Recombinant human endostatin (rhEndostatin, also known as Endostar or rh-Es) has been validated in published research for several clinical and preclinical applications, primarily centered on its anti-angiogenic and anti-tumor properties. The main applications supported by published studies include:

1. Cancer Therapy (Monotherapy and Combination)

• Non-Small Cell Lung Cancer (NSCLC): rhEndostatin is approved in China for first-line treatment of NSCLC and has demonstrated efficacy in improving response rates, clinical benefit rates, time to disease progression, and overall survival, especially when combined with chemotherapy (e.g., vinorelbine + cisplatin) or radiotherapy.
• Colorectal Cancer: Studies show that rhEndostatin, alone or in combination with radiotherapy, inhibits tumor growth and angiogenesis in colorectal cancer models.
• Other Solid Tumors: Clinical and preclinical studies have explored its use in gastric cancer, melanoma, breast cancer, and cervical cancer, with favorable safety and efficacy profiles.

2. Anti-Angiogenic Therapy

• rhEndostatin inhibits tumor angiogenesis by blocking the proliferation, migration, and survival of vascular endothelial cells, and by inducing their apoptosis.
• It downregulates VEGF expression and signaling, preventing VEGF from binding to its receptors and thereby blocking angiogenesis.

3. Combination with Chemotherapy and Radiotherapy

• rhEndostatin is frequently used in combination with chemotherapy (e.g., NP regimen: vinorelbine + cisplatin) or radiotherapy, showing enhanced therapeutic effects and improved outcomes compared to monotherapy.
• It can induce vascular normalization, which improves the delivery and efficacy of chemotherapeutic agents.

4. Metastasis and Tumor Progression

• rhEndostatin has been shown to reduce tumor metastasis (e.g., lung metastasis in breast cancer models) and inhibit primary tumor growth by targeting both angiogenesis and vasculogenic mimicry.

5. Immune Modulation

• Emerging evidence suggests that rhEndostatin may modulate the tumor immune microenvironment, including reprogramming tumor-associated macrophages (TAMs) and synergizing with immune checkpoint inhibitors.

6. Fibrosis Management

• Novel derivatives of endostatin (e.g., END55) have demonstrated anti-fibrotic efficacy in animal models and human tissue systems, suggesting potential applications in fibrotic disorders.

7. Diabetes and Metabolic Disorders

• Engineered variants of endostatin (e.g., ENP-9) have shown antihyperglycemic and antidiabetic effects in preclinical studies, indicating potential for metabolic disease applications.

8. Safety and Tolerability

• rhEndostatin has a favorable safety profile in clinical studies, with low toxicity and minimal side effects, even with long-term use.

Summary Table

These applications are supported by a broad range of peer-reviewed studies, meta-analyses, and clinical trials, establishing rhEndostatin as a versatile and promising therapeutic agent in oncology and beyond.

To reconstitute and prepare Recombinant Human Endostatin protein for cell culture experiments, follow these steps for optimal solubility, stability, and biological activity:

1. Centrifuge the vial before opening to ensure all lyophilized powder is at the bottom.

2. Reconstitution buffer:

   • Use sterile PBS (phosphate-buffered saline) or sterile distilled water as recommended by the product datasheet.
   • For enhanced stability and to prevent protein loss due to adsorption, especially at low concentrations, include a carrier protein such as 0.1% BSA (bovine serum albumin) in the buffer.

3. Reconstitution concentration:

   • A typical reconstitution concentration is 0.1–1.0 mg/mL.
   • Some protocols recommend 200 μg/mL in PBS.
   • Adjust the volume of buffer accordingly to achieve the desired concentration.

4. Dissolving the protein:

   • Add the calculated volume of buffer gently to the vial.
   • Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation (do not vortex or shake vigorously to avoid foaming and denaturation).

5. Aliquoting and storage:

   • Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles.
   • For short-term storage (up to 1 week), keep at 2–8°C.
   • For long-term storage, aliquot and freeze at –20°C to –80°C. Addition of a carrier protein (e.g., 0.1% BSA) and/or 5–50% glycerol can further stabilize the protein during freezing.

6. Working solution for cell culture:

   • Dilute the reconstituted stock to the desired working concentration using cell culture medium or buffer containing carrier protein to minimize adsorption losses.
   • Typical working concentrations for cell assays are in the range of 0.4–2 μg/mL, but this should be optimized for your specific application.

Key technical notes:

• If your experiment requires serum-free conditions, avoid animal-derived carrier proteins and consider using alternatives like trehalose.
• Always consult the specific product datasheet or Certificate of Analysis for any unique requirements or recommendations for your recombinant Endostatin preparation.

Summary Table:

Always use sterile technique throughout the process to maintain protein and cell culture integrity.