## 3 Biological Activities
### Antiproliferative Effect against Cancer Cells
In the same way, citral inhibits the proliferation of SK-MEL-147 and UACC-257 human melanoma cells. On the other hand, HaCaT human non-cancerous skin keratinocytes were more resistant to citral than NIH-3T3 murine non-cancerous fibroblasts. The effect of citral on NIH-3T3 was similar to the observed in B16F10 cancer cells, suggesting that citral exhibits greater selectivity against cancer cells in humans [9]. Citral is one of the major chemical constituents of the essential oil of _Lippiia citriodora_. [PERSON], [PERSON] [90] reported that citral is more effective than the essential oil of _Lcitriodora_ in inducing cytotoxicity and reducing the proliferation of HepG2 hepatocellular carcinoma cells, MCF-7 breast adenocarcinoma, THP-1 acute monocytic leukemia, A375 malignant melanoma, and Caco-2 cancer cells. Other research has shown that citral reduces the proliferation of KKU-M213 and HuCCA-1 cholangiocarcinoma cancer cells over time; however, the non-carcinogenic bile duct cell line MMNK-1 was more sensitive than HuCCA-1 cells [9]. [PERSON] et al. [94] reported that the effect of citral in inhibiting the cell proliferation of PC3 and PC3-M prostate cancer cells is similar to the effect showed by Cisplatin in these cells. However, Cisplatin was more cytotoxic against MRC-5 cells compared to citral. Morphological changes associated with apoptosis cell death, such as shrinkage and rounding, were observed after treating PC-3 and PC-3M cells with citral. In addition, the antiproliferative effect of citral against PaCa-2 pancreatic tumor cells and DeFew B-lymphoma cells has been reported [9]. In vitro studies have shown that citral inhibits the cancer cells colony formation and migration. [PERSON], [PERSON] [91] reported that citral affects the total number of colonies and the AGS cells' migration ability (\(p<0.001\) vs. control). Using Hoechst staining and DNA fragmentation assay, it was observed that citral induces chromatin condensation and the nuclear fragmentation of the cells in a dose-dependent manner, probably through apoptosis induction via ROS generation. Citral significantly reduced (\(p<0.001\) vs. control) the clonogenic formation of PC-3 cancer cells. [PERSON] [93] reported that citral promotes colony formation in KKU-M213 cells and decreases the survival fraction in HuCCA-1 and MMNK-1. These results did not directly correlate with the observed in vitro probably because the doses used in the clonogenicity tests were lower than the IC\({}_{50}\) values calculated for the in vitro experiments. On the other hand, citral reduced the colony formation of M624 cells more effectively than HaCaT cells [9]. Some mechanisms of action related to the antiproliferative effect of citral against cancer cells have been described. One of the reported mechanisms is the induction of cellular apoptosis. Apoptosis can be triggered through two main pathways: the intrinsic \begin{table}
\begin{tabular}{c c c c} \hline
**Compound/** & & **Effect** & **Ref.** \\
**Extract** & & & \\ \hline Citral & 0.25, 0.375, 0.5 mM & Caspase-3 activation in HaCaT (3 h) & [96] \\ Citral & 20 \(\mathrm{\SIUnitSymbolMicro g}\)/mL & Down-expression of HMGR, SREPB1, and ACC proteins in PC-3 (48 h) & [96] \\ Citral & 5, 10, and 20 \(\mathrm{\SIUnitSymbolMicro g}\)/mL & Up-expression of AMPeK in PC-3 (48 h) & [96] \\ Citral & 5, 10, and 20 \(\mathrm{\SIUnitSymbolMicro g}\)/mL & Down-expression of BCL-2 in PC-3 (48 h) & [96] \\ Citral & Not reported & and high expression of BAX proteins in PC-3 (48 h) & [91] \\ Citral & Not reported & mRNA upregulate in AGS (48 h): MAPK, Nf-kB, PI3K-Akt, p53, and other signaling pathways. Spliceosoma, apoptosis, and prostate cancer, among others. & [91] \\ Citral & Not reported & mRNA downregulate in AGS: Nf-kB, PI3K-Akt, p53, PPAR, among other signaling pathways. Cell cycle, fatty acid metabolism, and proteoglycans & [91] \\ Citral & 5, 10, and 20 \(\mathrm{\SIUnitSymbolMicro g}\)/mL & Down-expression of _HMCR_, _ACC_, _FASN_, and _SREPBI_ mRNAs in PC-3 & [94] \\ \hline \end{tabular}
\end{table}