Abstract
Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44+CD24-/low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment.
Highlights
Ginger (Zingiber officinale) is a well known herb consumed as a spice and food as well as widely used as herbal medicine for various ailments
Since 6-shogaol has been reported as a potent anticancer agent against various cancer cells, we have investigated its inhibitory effect on breast cancer cells and cancer stem cell-like spheroids
We found a significant increase in Light Chain3 (LC3)-II expression in the combination of 5 and 15 μM of 6-shogaol with chloroquine (3.3 and 3.43 fold increase) when compared with the treatment of 6-shogaol alone (1.8 and 2.1 fold) (Fig 6B)
Summary
Ginger (Zingiber officinale) is a well known herb consumed as a spice and food as well as widely used as herbal medicine for various ailments. Cancer cells can be made to grow in the form of spheroids These spheroid-forming cells exhibit altered cell surface markers when compared to cells grown in monolayer culture and have been shown to possess stem-cell like properties [10, 13]. These spheroids have been used in a number of studies to determine the in vitro and in vivo characteristics of cancer stem cells as well as to assess the inhibitory activity of cytotoxic compounds against cancer stem cells [11, 14, 15]. Investigation of the death mechanism shows that autophagy is a predominant mode of cell death caused by 6-shogaol in breast cancer cells
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