Naturally occurring phenethyl isothiocyanate inhibits gastric cancer cell growth by disruption of microtubules

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The dietary plant phytochemical PEITC is thought to contribute to chemoprevention against several types of human cancer diseases alongside with other plant phytochemicals enriched in a diet with cruciferous vegetables. Moreover, PEITC has also been suggested for clinical treatment of cancer as it may cure resistance to cancer drugs and thus sensitize cancer cells to these drugs²². It is therefore important to understand the underlying mechanisms upon the PEITC-entry to a cancer cell. In the present study we showed a disintegration of microtubular filaments in gastric cancer cell line Kato-III as a mechanism presumably contributing to the subsequent cell cycle arrest and apoptosis induced by PEITC. Previous studies with ITCs and stomach cancer have shown an inverted correlation between intake of ITC-rich diet and risk of cancer⁷. Furthermore, the broccoli-derived sulforaphane was reported to eradicate the gastric cancer-related bacterium H. pylori from a gastric cancer cell line and presumably reduced the cell count of these bacteria in the stomach of human patients^{18, 23}. Prevention of chemically induced gastric cancer in mice by SFN was linked to the nrf2-gene as nrf2 knock out mice did not respond equally strong in cancer prevention to an SFN-rich diet¹⁸. To the best our knowledge, no in vivo experiments on gastric cancer with PEITC have been reported so far, which we in parallel to this study is currently undertaking. However, in in vitro studies, PEITC has been shown to attenuate cell migration and invasion of human gastric cancer AGS cell line¹⁷. Suppression of MAPK- and NFkB signalling pathways were pointed out as key underlying factors. Similar findings were made when another aromatic ITC, benzyl ITC, was applied to AGS cells²⁴. Although our findings are the first to show the loss of microtubular filaments in gastric cancer cells treated with PEITC, in lung cancer cells, Mi and colleagues identified alpha- and beta tubulins, monomers of microtubuli as binding targets for PEITC¹². Moreover, in bladder cancer cells, binding of the aliphatic allyl ITC to alpha- and beta tubulin led to subsequent ubiquitination and degradation of tubulins as well as onset of a mitotic cell cycle arrest and ultimately apoptosis²¹. In prostate cancer cells, PEITC was shown to induce G2/M cell cycle arrest as well as downregulation of gene expression of alpha- and beta tubulins²⁵. In breast cancer cells, SFN was shown to suppress the dynamic instability of microtubules leading to mitotic arrest of these cells²⁶. Taken together we suggest that PEITC binds to alpha- and beta tubulins with the subsequent degradation of these monomers leading to a deformation of microtubular filaments which in turn contributes to accumulation of cells in G₂/M-phase and apoptosis in gastric cancer cell line Kato-III. Upon entering a cell, PEITC inevitably binds to the abundantly present redox mediator GSH. Binding and conjugation with GSH leads to a decrease in the intracellular GSH-pool and secondary effects including generation of ROS. It has been suggested that this mechanism is essential in the selective toxicity of PEITC in cancerous cells compared to their normal equivalents²⁷. Cell line MKN74 tested in the present study displayed a lower sensitivity to PEITC when proliferation was assayed. Furthermore, these cells showed a weak response in GSH-reduction, increase of apoptotic cells, and increase in caspase-3 activity. When testing higher concentrations than those presented here, we observed high amount of cell death and could not obtain reproducible data. Different sensitivity among individual cell lines has also been observed in human breast cancer cells in which a comparison of two cell lines revealed the one to yield a higher IC₅₀-value, lower increase in apoptotic cells and a lower decrease in GSH-content following PEITC-treatment²⁸. These observations were accredited the elevated basal level of NRF2 in the lesser sensitive cell line, as an elevated level of NRF2 also results in and increase in GSH-level rendering the cells more resistant to PEITC. Important features of cancerous cells are the elevated level of ROS²⁷, and the ability to promote NRF2-dependent ROS-detoxification²⁹, which again points to the importance of the basal GSH-level which presumably may vary with cell types. Thus, the reduced sensitivity in MKN74 cells might be explained through an elevated basal level of GSH-content in consistency with no elevation of ROS-levels yet a weak but significant reduction in GSH-content following PEITC-treatment. In conclusion, the present study demonstrates PEITC as a potential inhibitor of gastric cancer cell growth, and presents new insight into the underlying molecular mechanisms.