Dr Weeks’ Comment: Many of us grew up being treated with Ginger Ale which, as is the case with many spices and bitters in cocktails, is a medicine. That gin and tonic has quinine in it which helped the Brits survive malaria in tropical climes. Now we are reminded that ginger (read below) kills cancer stem cells – more powerfully than many chemo drugs ! Grated ginger also helps with motion sickness!
6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death
Authors: Anasuya Ray, Smreti Vasudevan, Suparna Sengupta SEPT 2015
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 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.
Cancer stem cells play a very important role in cancer development and progression. The concept of stem cell origin of cancer has been supported by observations that certain subpopulations (only 0.2–1%) of cancer cells have stem cell-like properties, such as the ability to self renew, continuous differentiation and an overall innate resistance to conventional chemotherapeutic agents . These chemo-resistant, self-renewing, tumorigenic sub-population of cells defined as cancer stem cells (CSCs) play crucial roles in cancer recurrence. CSCs have been identified in various solid tumors including breast, ovarian, head and neck, pancreas, and colon cancer [9, 10]. Earlier studies demonstrated that the signaling pathways such as Wnt/β-catenin, Notch and Hedgehog pathways regulate the growth of cancer stem cells [11, 12]. Therefore, targeting these pathways is considered to be a useful strategy to inhibit cancer stem cell regeneration.
Cancer stem cells pose serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. To add into the misery, very few chemotherapeutic compounds show promise to kill these cells. Several researchers have shown that cancer stem cells are resistant to paclitaxel, doxorubicin, 5-fluorouracil, and platinum drugs [8, 16]. CSCs are thus an almost unreachable population in tumours for chemotherapy. Therefore any compound, that shows promise towards cancer stem cells, is a highly desirable step towards cancer treatment and should be followed up for further development.
Dietary compounds are welcome options for human diseases due to their time-tested acceptability by human bodies. Curcumin , quercetine , garlic products , ginger products  are some of them which show very high potency in many human diseases including cancer. In this study we have found that the ginger product 6-shogaol was effective in breast cancer cells in monolayer culture and spheroid culture in comparable concentrations and in conditions where taxol, even though highly effective in monolayer cells, was completely ineffective (Table 1). The effective concentrations of 6-shogaol were also safe to noncancerous cells as shown from the significantly higher IC50 values in case of HEK 293 and HaCaT cells even after 6 days (Table 1 and Fig 2C). The breast cancer spheroids generated by us under specific culture conditions showed altered levels of the breast CSC markers CD44+CD24–/low. These cells also showed secondary sphere forming capacity. 6-shogaol could drastically reduce the percentage of cells expressing the markers as well as the secondary sphere formation showing that it really could target the cancer stem cell like cells.
Cell cycle analysis of 6-shogaol treated cells showed G2/M arrest in both monolayer and spheroids. This is expected as 6-shogaol has been shown to interact with microtubules causing a mitotic block . However, the percentage of apoptotic cells induced in MCF-7 monolayer cells was rather low as determined by different apoptotic assays. Further, apoptosis occurred only by a 2–3 times higher IC50 concentration of 6-shogaol. In spheroid culture, we couldn’t see considerable apoptotic cells even after 96 hours at a concentration slightly higher than the IC50. However, massive cell death at that concentration and beyond indicated other mechanisms of cell death.
Autophagy is considered as a basic survival mechanism of cells under stress. However, many reports show that autophagy is induced with a death trigger. Debate is still going on whether autophagy is another cell death mechanism besides apoptosis or cells die with autophagy using it as an effort to survive . Induction of autophagy in 6-shogaol treated breast cancer cells was proved by cytoplasmic vacuole formation as well as the recruitment and cleavage of the microtubule associated protein Light Chain3 (LC3). As apoptosis was found to be a minor mode of cell death in these cells, the role of autophagy in 6-shogaol treatment needed further perusal. Our kinetic study of LC3 accumulation as well as combination studies with the autophagy inhibitor chloroquine (CQ) for LC3 accumulation and cell death convincingly proved that autophagy was the predominant cell death mechanism induced by 6-shogaol in breast cancer cells. A former report also showed that 6-shogaol was able to cause autophagy in HNSCLC A-549 cells and autophagy was the death mechanism in those cells in absence of apoptosis .
Several earlier studies have shown that dietary compounds which are used as anticancer agents, exhibited their interference with self-renewal pathways. Sulforaphane, curcumin and piperine inhibited Wnt pathway in breast cancer stem cells [14, 17]. Curcumin showed Notch pathway inhibition in esophageal cancer cells . Notch signaling plays an important role in the development of mammary gland and is activated in stem cell self-renewal and differentiation [12, 39]. Many solid tumours including breast tumour also show deregulated Notch signaling [30, 40, 41]. 6-shogaol was found to interfere with the Notch pathway. Further, in our study, combination of the γ-secretase inhibitor DAPT and 6-shogaol resulted in further reduction of the expression of Notch and its target proteins (Fig 8B) when compared to the reduction by either DAPT or 6-shogaol alone. The combination also gave rise to lesser number of primary and secondary spheres than those generated from the individual treatments (Fig 8C). This indicates that even though both DAPT and 6-shogaol inhibited γ-secretase, their action was not competitive. γ-secretase mediated downregulation of Notch signaling followed by reduction in Notch1 downstream products like Hes1 and cyclin D1 by 6-shogaol thus explain its efficacy in breast cancer spheroid cells.
An earlier pharmacokinetic study  of 6-shogaol and three gingerols on healthy humans reported that after p.o. (orally administered) dosing, all these compounds were absorbed. These compounds were later found to be metabolized as glucuronide and sulfate conjugates. No serious adverse effects were seen on those persons after ingestion of a single dose of 2 g of standardized ginger extract. A former report also showed that 6-shogaol was not toxic to normal cells like MRC5, NP69, HaCaT and HMEC . Our results on immortalized noncancerous cells thus supported that 6-shogaol did not have potent cytotoxicity to normal cells.
Our study shows the efficacy of 6-shogaol on both breast cancer monolayer cells and spheroids (a scheme shown in Fig 9) by interfering with stem cell self-renewal pathway. This report thus places it as a promising therapeutic agent which should be further followed up for breast cancer treatment.