A team of researchers from Spain and Sweden recently examined the effects of rosemary (Rosmarinus officinalis L.) on colon cancer cell proliferation. It may seem like a feeble treatment for something as serious as cancer, but Valdés et al. explain that rosemary has documented health benefits, with its medicinal uses dating back thousands of years. The high antioxidant activity in rosemary comes from polyphenol compounds acting in different molecular levels to change protein expression. Ultimately, the team found that the rosemary polyphenols did induce proteomic changes in the colon cancer cell line.1
During their analysis, the team focused on three target areas:
- Effects on the protein amplitude and kinetics
- Proteomic differences between the cytostatic and cytotoxic effects
- Antiproliferative mechanisms induced by rosemary polyphenols
The researchers grew HT-29 colon cancer cells and treated them with different concentrations of a polyphenol-enriched rosemary extract over time. The amount of rosemary ranged from none to 60 μg/ml. For controls, they incubated the HT-29 cells with 0.2% (v/v) dimethyl sulfoxide for the same time periods as the experimental group. After six hours, 12 hours, and 24 hours of incubation time, the team removed the culture media added a complete media. They analyzed the cells at 24 hours and 48 hours.
Valdés et al. chose a proteomics strategy based on nano liquid chromatography−tandem mass spectrometry (nLC-MS/MS). They also employed stable isotope dimethyl labeling using “light” and “medium” labels for control and rosemary-treated cells, respectively. For the analysis, they used an EASY-nLC II system (Thermo Scientific) coupled via a nanoelectrospray ionization ion source to an LTQ Velos Pro mass spectrometer (Thermo Scientific).
The researchers found that six hours of incubation time was enough to induce cytostatic effects. Upon withdrawal of the media, they saw that the effects diminished and cells partially recovered. In contrast, longer exposures (12 hours and 24 hours) to the same extract concentration had stronger cytotoxic effects, indicating that expression changes occurred in a time- and concentration-dependent manner.
The team identified and quantified 1,909 and 698 proteins, respectively, in cell extracts. Using a microarray, the investigators identified 2,211 mRNAs (about 5% of the total genes on the array) as differentially expressed (false discovery rate < 5%) in HT-29. Cross-referencing data revealed that the transcriptomics data overlapped 89.9% of the proteomic data, representing 771 proteins. Of these, 15.2% and 8.9% of proteins revealed alterations at the protein and mRNA levels, respectively. The vast majority of altered proteins activated the Nrf2 transcription factor. The higher extract concentrations induced two relevant transcription factors (Atf4 and Xbp1) related to unfolded protein response induction.
The authors consider that inducing stress with Nrf2-inducing agents, such as rosemary polyphenols, unlocks new possibilities to treat cancer.
1. Valdés, A.J., et al. (2016) “Comprehensive proteomic study of the antiproliferative activity of a polyphenol-enriched rosemary extract on colon cancer cells using nanoliquid chromatography-orbitrap MS/MS,” Journal of Proteome Research, 15(6) (pp. 1971–1985), doi: 10.1021/acs.jproteome.6b00154.