Background: Citrus bioactive compounds, as active anticancer agents, have been under
focus by several studies worldwide. However, the underlying genes responsible for the anticancer
potential have not been sufficiently highlighted.
Objectives: The current study investigated the gene expression profile of hepatocellular carcinoma,
HepG2, cells after treatment with Limonene.
Methods: The concentration that killed 50% of HepG2 cells was used to elucidate the genetic
mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry
and confocal fluorescence microscope. Two of the pro-apoptotic events, caspase-3 activation and
phosphatidylserine translocation were manifested by confocal fluorescence microscopy. Highthroughput
real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families
related to the cancer development.
Results: In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to
89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene-
driven differential gene expression of HepG2 cells in 15 different gene families. Limonene
was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively.
The affected gene families, from the most to the least affected, were apoptosis induction, signal
transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage
repair, and cell cycle proteins.
Conclusion: The current study reveals that limonene could be a promising, cheap, and effective
anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer
drug development. Further research is needed to confirm the current findings and to examine the
anticancer potential of limonene along with underlying mechanisms on different cell lines.