Potential Anticancer Effects of Limonene Associated with Reactive Oxygen Species Generation, Apoptosis Induction, and NF-κB Modulation in Papillary Renal Cell Carcinoma: A Preliminary Study.

BACKGROUND

Tumor recurrence and progression occur in many patients with renal cell carcinoma, highlighting a clinical demand for safe and effective treatment options, particularly by employing plant-derived bioactive compounds. Limonene, an aromatic monoterpene commonly found in citrus peels, has been shown to possess anticancer effects in malignancies such as prostate and bladder cancers.

OBJECTIVES

This study aimed to explore the potential anticancer effects of limonene on ACHN papillary renal cancer cells (pRCC).

METHODS

The viability of ACHN, human embryonic kidney (HEK293), and human dermal fibroblast (HDF) cells treated with limonene was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Cellular migration, colony formation, 3D spheroid development, morphological alterations, and caspase 3/7 activity were evaluated in vitro. Reactive oxygen species (ROS) levels, apoptosis, and cell cycle alterations were analyzed by flow cytometry. The mRNA levels of BAX, BCL2, E-cadherin, N-cadherin, Caspase-3, Caspase-9, NF-κB, Akt1, MMP-2, MMP-9, mTOR, TNF-α, COX-2, and IL-6 were quantified using quantitative polymerase chain reaction. NF-κB protein levels and IκB phosphorylation were assessed by Western blot. Nuclear NF-κB levels were measured by ELISA.

RESULTS

The viability of ACHN cells was reduced by limonene in a dose- and time-dependent manner, with comparatively limited effects on HEK293 and HDF cells. Limonene significantly reduced migration, colony formation, and spheroid growth in ACHN cells. Furthermore, limonene elevated ROS levels and increased apoptosis in ACHN cells without affecting cell cycle progression. After limonene exposure, mRNA expression of Akt1, mTOR, NF-κB, MMP-2, N-cadherin, BCL2, TNF-α, COX-2, and IL-6 decreased, whereas BAX and E-cadherin expression increased. Caspase-3 and Caspase-9 mRNA levels were not significantly altered, whereas caspase 3/7 activity increased. Limonene also reduced NF-κB protein levels, IκB phosphorylation, and NF-κB nuclear translocation.

CONCLUSIONS

These preliminary in vitro findings support further investigation of limonene as a therapeutic candidate in additional pRCC models.