Cytotoxicity of Cucurbitacin E From Citrullus Colocynthis Against Multidrug-Resistant Cancer Cells

Phytomedicine. 2019 Sep;62:152945. doi: 10.1016/j.phymed.2019.152945. Epub 2019 May 2.

Abstract

Background: Cucurbitacin E (CuE) is an oxygenated tetracyclic triterpenoid isolated from the fruits of Citrullus colocynthis (L.) Schrad.

Purpose: This study outlines CuE's cytotoxic activity against drug-resistant tumor cell lines. Three members of ABC transporters superfamily, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and ABCB5 were investigated, whose overexpression in tumors is tightly linked to multidrug resistance. Further factors of drug resistance studied were the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR).

Methods: Cytotoxicity assays (resazurin assays) were used to investigate the activity of Citrullus colocynthis and CuE towards multidrug resistant cancer cells. Molecular docking (In silico) has been carried out to explore the CuE's mode of binding to ABC transporters (P-gp, BCRP and ABCB5). The visualization of doxorubicin uptake was done by a Spinning Disc Confocal Microscope. The assessment of proteins expression was done by western blotting analysis. COMPARE and hierarchical cluster analyses were applied to identify, which genes correlate with sensitivity or resistance to cucurbitacins (CuA, CuB, CuE, CuD, CuI, and CuK).

Results: Multidrug-resistant cells overexpressing P-gp or BCRP were cross-resistant to CuE. By contrast, TP53 knock-out cells were sensitive to CuE. Remarkably, resistant cells transfected with oncogenic ΔEGFR or ABCB5 were hypersensitive (collateral sensitive) to CuE. In silico analyses demonstrated that CuE is a substrate for P-gp and BCRP. Immunoblot analyses highlighted that CuE targeted EGFR and silenced its downstream signaling cascades. The most striking result that emerged from the doxorubicin uptake by ABCB5 overexpressing cells is that CuE is an effective inhibitor for ABCB5 transporter when compared with verapamil. The COMPARE analyses of transcriptome-wide expression profiles of tumor cell lines of the NCI identified common genes involved in cell cycle regulation, cellular adhesion and intracellular communication for different cucurbitacins.

Conclusion: CuE represents a potential therapeutic candidate for the treatment of certain types of refractory tumors. To best of our knowledge, this is the first time to identify CuE and verapamil as inhibitors for ABCB5 transporter.

Keywords: Cancer; Citrullus colocynthis; Drug resistance; Microarray; Traditional herbal medicine.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B / metabolism
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / chemistry
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • ATP Binding Cassette Transporter, Subfamily G, Member 2 / genetics
  • ATP Binding Cassette Transporter, Subfamily G, Member 2 / metabolism
  • Antineoplastic Agents, Phytogenic / chemistry
  • Antineoplastic Agents, Phytogenic / pharmacology*
  • Cell Line, Tumor
  • Citrullus colocynthis / chemistry*
  • Doxorubicin / pharmacology
  • Drug Resistance, Neoplasm / drug effects
  • ErbB Receptors / genetics
  • Gene Expression Regulation, Neoplastic / drug effects
  • Gene Knockout Techniques
  • Humans
  • Leukemia / drug therapy*
  • Leukemia / metabolism
  • Leukemia / pathology
  • Molecular Docking Simulation
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism
  • Triterpenes / chemistry
  • Triterpenes / metabolism
  • Triterpenes / pharmacology*
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • ABCB5 protein, human
  • ABCG2 protein, human
  • ATP Binding Cassette Transporter, Subfamily B
  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • Antineoplastic Agents, Phytogenic
  • Neoplasm Proteins
  • TP53 protein, human
  • Triterpenes
  • Tumor Suppressor Protein p53
  • Doxorubicin
  • EGFR protein, human
  • ErbB Receptors
  • cucurbitacin E