Investigation of the Effect of 5‑Aza‑2’‑Deoxycytidine in Comparison to and in Combination with Trichostatin A on p16INK4a, p14ARF, p15INK4b Gene Expression, Cell Growth Inhibition and Apoptosis Induction in Colon Cancer Caco‑2 Cell Line

Masumeh Sanaei, Fraidoon Kavoosi, Vahid Ghasemzadeh

Abstract


Background: The cell cycle is divided into four phases, G1, G2, S, and M phase. The mammalian cell cycle is controlled and governed by the kinase complexes including cyclin and the cyclin‑dependent kinase (CDK), cyclin‑CDK complexes. The activity of the complexes is regulated by cyclin‑dependent kinase inhibitors (CDKIs), the INK4, and the CDK interacting protein/kinase inhibitory protein (CIP/KIP) families. Promoter hypermethylation and histone deacetylation of CDKIs have been reported in several cancers. These changes can be reversed by DNA demethylating agents, such as decitabine, 5‑Aza‑2′‑deoxycytidine (5‑Aza‑CdR), and histone deacetylase inhibitors (HDACIs), such as trichostatin A. Previously, we reported the effect of 5‑Aza‑CdR and trichostatin A (TSA) on hepatocellular carcinoma (HCC). The present study aimed to investigate the effect of 5‑Aza‑CdR in comparison to and in combination with trichostatin A on p16INK4a, p14ARF, p15INK4b genes expression, cell growth inhibition and apoptosis induction in colon cancer Caco‑2 cell line.

Methods: The Caco‑2 cells were cultured and treated with 5‑Aza‑CdR and TSA (alone and combined). The cell viability, apoptosis, and relative gene expression were determined by MTT assay, flow cytometry, and real‑time quantitative reverse transcription‑polymerase chain reaction (qRT‑PCR), respectively.

Results: Both compounds inhibited cell growth, induced apoptosis, and up‑regulated the p16INK4a, p14ARF, p15INK4b gene significantly. The TSA had a more significant effect in comparison to 5‑Aza‑CdR. Furthermore, maximal apoptosis and up‑regulation were observed with combined treatment.

Conclusions: our finding indicated that 5‑Aza‑CdR and TSA can epigenetically re‑activate the p16INK4a, p14ARF, p15INK4b gene resulting in cell growth inhibition and apoptosis induction in colon cancer.


Keywords


Colonic neoplasms; cyclin‑dependent kinase inhibitors; decitabine; trichostatin A

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