**7. The Metabolite Hypothesis—A Common Mechanism for Cancer Prevention**

The unstable nature of flavonoids, the rapid hydrolysis of aspirin in the gut and the reports on the cancer-preventive potential of their degraded products through inhibition of cancer cell growth collectively suggest that the HBAs generated from flavonoids and aspirin may be key contributors to their cancer prevention properties. Although the parent compounds may directly contribute to the observed chemopreventive effects through other pathways already reported in literature (Figure 1), we suggest that the contribution of HBAs should also be taken into account. Unabsorbed flavonoids and aspirin/salicylic acid may act as substrates for the gut microbial enzymes to convert them into simpler, pharmacologically active HBAs, like 2,3-DHBA, 2,5-DHBA from aspirin and 3,4-DHBA, 3,4,5-THBA and 2,4,6-THBA from flavonoids. The generation of such HBAs in the gut compel us to propose a central role for these molecules in cancer prevention by aspirin, flavonoids and the diet. As these HBAs are metabolites generated through biotransformation in the body and are also secondary metabolites in plants with the capacity to inhibit cancer cell growth, we would like to refer to this hypothesis as "metabolite hypothesis". A model depicting the convergence of the pathways generating HBAs from aspirin, flavonoids and diet through microbial/host enzymes is shown in Figure 3. The chemopreventive ability of aspirin and flavonoids have been established through many epidemiological studies and as the exact mechanisms of chemoprevention for these compounds have not been clearly established, we are suggesting through this review, that HBAs may be contributing to their chemopreventive actions. Though the data that was published previously on the ability of HBAs to prevent cancer cell growth were performed with cancerous cell lines, and therefore are more reflective of therapy than prevention, we believe that these observations can be extrapolated to cancer prevention. However, the extent to which HBAs are generated from these compounds and their overall contribution to the cancer prevention potential of the parent compounds require further investigation. Cancer prevention by HBAs is relatively underexplored, and further investigations are required to identify potential targets (intracellular vs. extracellular), their uptake mechanisms by cells, signaling pathways and target gene expression. Such studies would have tremendous implications in developing new strategies for cancer prevention.

**Figure 3.** Metabolite hypothesis: Model depicting convergence of the pathways generating HBAs from parent compounds through host/microbial enzymes. We propose that actions of HBAs, generated through biotransformation of aspirin and flavonoids, retard rate of cell proliferation. This would provide an opportunity for (i) immune surveillance, leading to the destruction of cancer cells, or (ii) DNA repair in cells containing damaged DNA, providing genetic stability, both of which are important steps in the prevention of cancer. While 2,4,6-THBA is likely to retard cell proliferation through CDK inhibition and upregulation of p21Cip1 and p27Kip1, the exact mechanisms of cell growth inhibition by other HBAs is still not understood [57,63]. EGCG—Epigallocatechin gallate, C3G—Cyanidin-3-glucoside.

#### **8. Conclusions**

It has been established over the course of time that increased consumption of fruits and vegetables has health benefits, making Hippocrates' dictum "Let food be your medicine and medicine your food" all the more significant. Identification of polyphenols and phenolic acids commonly found as a component of the diet has now led to the generation of nutraceutical and/or pharmaceutical compounds that have revolutionized the health sector. Considering the increasing incidence of CRC worldwide, it is now more important than ever to come up with viable preventive strategies against CRC. We believe that HBAs merit further studies to understand their role in cancer prevention as the proposed mechanism (metabolite hypothesis) involving HBAs is a simple and tenable explanation for CRC prevention by both aspirin and flavonoids. Since intestinal epithelial cells are the first to get exposed to HBAs following their consumption or metabolism (from aspirin/flavonoids), in our view CRC prevention is likely to be a local effect. The colorectal tissues may thus have an anatomical advantage as they are the first to get exposed to these HBAs, making them preferred targets, while those HBAs absorbed into circulation may affect cancer development in other tissues as well. We believe that effective cancer prevention requires the partnership between the parent compounds and the right microbial species responsible for HBA generation. We also propose that the inter-individual variability previously observed in many epidemiological studies [8,17,27] could be attributed to the lack of an appropriate microbial ecology necessary for their degradation. Therefore, a strategy involving supplementation of the diet with appropriate probiotics and aspirin/flavonoids, or directly consuming HBAs may prove useful in CRC prevention.

**Author Contributions:** R.S. and G.J.B. wrote the initial draft of the manuscript, D.R.K. and J.P. were involved in discussion, editing and revising of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by funds from the Office of Research and the Department of Pharmaceutical Sciences, South Dakota State University.

**Acknowledgments:** Authors acknowledge Chaitanya Valiveti for useful discussions.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
