An in vitro cytotoxicity of glufosfamide in HepG2 cells relative to its nonconjugated counterpart

Doaa E Ahmed; Fatma B Rashidi; Heba K Abdelhakim; Amr S Mohamed; Hossam M M Arafa

doi:10.1186/s43046-021-00080-6

An in vitro cytotoxicity of glufosfamide in HepG2 cells relative to its nonconjugated counterpart

J Egypt Natl Canc Inst. 2021 Aug 23;33(1):22. doi: 10.1186/s43046-021-00080-6.

Authors

Doaa E Ahmed¹, Fatma B Rashidi², Heba K Abdelhakim³, Amr S Mohamed³, Hossam M M Arafa⁴

Affiliations

¹ Department of Biochemistry, Misr University for Science & Technology (MUST), Giza, Egypt.
² Biochemistry Lab.Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt. fatmabaiuomy@yahoo.com.
³ Biochemistry Lab.Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt.
⁴ Department of Pharmacology and Toxicology, Faculty of Pharmacy Ahram Canadian University, Giza, 267119, Egypt.

PMID: 34423383
DOI: 10.1186/s43046-021-00080-6

Abstract

Background: Glufosfamide (β-D-glucosylisophosphoramide mustard, GLU) is an alkylating cytotoxic agent in which ifosforamide mustard (IPM) is glycosidically linked to the β-D-glucose molecule. GLU exerted its cytotoxic effect as a targeted chemotherapy. Although, its cytotoxic efficacy in a number of cell lines, there were no experimental or clinical data available on the oncolytic effect of oxazaphosphorine drugs in hepatocellular carcinoma. Therefore, the main objective of the current study is to assess the cytotoxic potential of GLU for the first time in the hepatocellular carcinoma HepG2 cell line model.

Methods: Cytotoxicity was assayed by the MTT method, and half-maximal inhibitory concentration (IC₅₀) was calculated. Flow cytometric analysis of apoptosis frequencies was measured by using Annexin V/PI double stain, an immunocytochemical assay of caspase-9, visualization of caspase-3, and Bcl2 gene expression were undertaken as apoptotic markers. Mitochondrial membrane potential was measured using the potentiometric dye; JC-1, as a clue for early apoptosis as well as ATP production, was measured by the luciferase-chemiluminescence assay.

Results: Glufosfamide induced cytotoxicity in HepG2 cells in a concentration- and time-dependent manner. The IC₅₀ values for glufosfamide were significantly lower compared to ifosfamide. The frequency of apoptosis was much higher for glufosfamide than that of ifosfamide. The contents of caspase-9 and caspase-3 were elevated following exposure to GLU more than IFO. The anti-apoptotic Bcl2 gene expression, the mitochondrial membrane potential, and the cellular ATP levels were significantly decreased than in case of ifosfamide.

Conclusions: The current study reported for the first time cytotoxicity activity of glufosfamide in HepG2 cells in vitro. The obtained results confirmed the higher oncolytic activity of glufosfamide than its aglycone ifosfamide. The generated data warrants further elucidations by in vivo study.

Keywords: Bcl-2; Caspase-9; Glufosfamide HepG2 cytotoxicity; Ifofosfamide (IFO); Mitochondrial membrane potential.

MeSH terms

Glucose / analogs & derivatives
Hep G2 Cells
Humans
Ifosfamide* / analogs & derivatives
Liver Neoplasms* / drug therapy
Liver Neoplasms* / genetics

Substances

beta-D-glucosylisophosphoramide mustard
Glucose
Ifosfamide