Characterization of in vivo chemoresistant human hepatocellular carcinoma cells with transendothelial differentiation capacities.

Abstract

BACKGROUND:

Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments a better understanding of biochemical changes in the resistant tumors is needed. In this study, we focus on the characterization of in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft model.

METHODS:

SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. Tumors were evaluated by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Cell lines of these tumors were analyzed by qRT-PCR and in endothelial transdifferentiation studies on matrigel.

RESULTS:

HUH-REISO cells, although slightly more sensitive against activated CPA in vitro than parental HUH-7 cells, fully retained their in vivo CPA chemoresistance upon xenografting into SCID mice. Histochemical analysis of HUH-REISO tumors in comparison to parental HUH-7 cells and passaged HUH-PAS cells (in vivo passaged without chemotherapeutic pressure) revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO. In transdifferentiation studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. A comparative study on stemness and plasticity markers revealed upregulation of Thy-1, Oct-4, Sox-2 and Nanog in resistant xenografts. Under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression.

CONCLUSIONS:

Chemoresistance of HUH-REISO was not manifested under standard in vitro but under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of transdifferentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.

• Authors: Christian Marfels, Miriam Hoehn, Ernst Wagner, Michael Günther
• Journal: BMC cancer (BMC Cancer) Vol. 13 Pg. 176 (Apr 02 2013) ISSN: 1471-2407 [Electronic] England
• PMID: 23547746 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antineoplastic Agents
• Basic Helix-Loop-Helix Transcription Factors
• Homeodomain Proteins
• Isoenzymes
• NANOG protein, human
• NOTCH1 protein, human
• Nanog Homeobox Protein
• Octamer Transcription Factor-3
• Receptor, Notch1
• SOXB1 Transcription Factors
• Thy-1 Antigens
• Transcription Factor HES-1
• HES1 protein, human
• Aldehyde Dehydrogenase 1 Family
• ALDH1A1 protein, human
• ALDH1A1 protein, mouse
• Retinal Dehydrogenase

Topics

• Administration, Metronomic
• Aldehyde Dehydrogenase 1 Family
• Animals
• Antineoplastic Agents (pharmacology, therapeutic use)
• Basic Helix-Loop-Helix Transcription Factors (genetics)
• Carcinoma, Hepatocellular (drug therapy, genetics, pathology)
• Cell Line, Tumor
• Cell Transdifferentiation
• Disease Models, Animal
• Drug Resistance, Neoplasm
• Endothelial Cells (pathology)
• Female
• Gene Expression Profiling
• Homeodomain Proteins (genetics)
• Humans
• Isoenzymes (genetics)
• Liver Neoplasms (drug therapy, genetics, pathology)
• Male
• Mice
• Nanog Homeobox Protein
• Neovascularization, Pathologic (drug therapy, metabolism)
• Octamer Transcription Factor-3 (genetics)
• Receptor, Notch1 (genetics)
• Retinal Dehydrogenase (genetics)
• SOXB1 Transcription Factors (genetics)
• Spheroids, Cellular
• Thy-1 Antigens (genetics)
• Transcription Factor HES-1
• Tumor Cells, Cultured
• Xenograft Model Antitumor Assays