Lung Cancer Chemoprevention of Ginsenosides is Mediated by the Glycosidases Activities of the A/J Mouse Intestinal Microbiome Defined by Pyrosequencing

[Purpose] Red ginseng extract (RGE) has been reported to possess non-organ-specific preventative effects against several types of cancer, including lung cancer. The ginsenosides responsible for the activity of RGE are poorly defined as primary ginsenosides are inactive. Since most of the primary ginsenosides need the action of bacterial glycosidases to become active one, an important question is if functional activity of bacterial glycosidases would be altered following RGE administration. A more intriguing question is if and how RGE treatment can affect the growth of certain bacteria population in the intestinal microbiome, and whether alteration of such bacteria population would impact the chemoprevention efficacy of RGE. Therefore, the central hypothesis of this thesis is that the lung cancer chemoprevention of ginsenosides is mediated by the glycosidases activites of the A/J mouse intestinal microbiome defined by pyrosequencing. To test the central hypothesis, three specific aims are i) to characterize microbiota dependent metabolism of RGE in A/J mouse fecal lysate; ii) to determine the impacts of RGE administration on bacterial glycosidase activity and intestinal microbiome in A/J mouse; iii) to purifiy and identify bacterial glycosidase(s) from A/J mouse feces that catalyze the rate limiting step in the production of ginesnoside Compound K (one of the most active ginsenosides in RGE). [Methods] The kinetics of microbiota mediated biotransformation of ginesnosides was characterized and kinetic parameters (metabolite formation rates) were determined. The anti-proliferative activity of ginsenosides was tested using the mouse lung cancer LM1 cells. Permeabilities of ginesnosides were also evaluated in Caco-2 cell monolayers. The dose dependent enzymatic functions was tested by giving RGE orally at three doses (daily oral gavage at 5, 50, and 500 mg/kg respectively). The impacts of RGE administration on bacterial glycosidase and intestinal microbiome of A/J mice were evaluated by giving RGE daily to A/J mice for 7 days followed by measuring kinetic parameters (metabolite formation rates), diversity metrics (α diversity and β diversity) and relative abundance of bacteria in the gut microbiome. Bacterial glycosidases were enriched from A/J mouse feces by a classic protein chemistry approach. The identity of the enzymes was examined by LC-MS/MS analysis followed by gene synthesis, molecular cloning, and expression of the enzymes. The functional activity of the enzymes against ginsenoside Rd was tested. [Results] I) Compound K exhibited higher anti-proliferative activity (IC50 ~13 µg/mL) and better permeability (1 ×10-6 cm/s) than primary ginsenosides. Primary ginsenoside Rb1 was converted to Rd, F2, and then Compound K by A/J mouse fecal lysate in a stepwise fashion. Formation of F2 from Rd (metabolite formation rate 0.09 ± 0.003, 0.09 ± 0.01 nmol/min/mg at 20, 5 μM substrate concentration respectively) was the slowest step in the biotransformation of Rb1 to Compound K. II) Bacterial glycosidase activity in response to RGE treatment exhibited a dose dependent manner and the optimal dose of RGE was found to be 50 mg/kg. Oral administration of RGE at 50 mg/kg for 7 days significantly enhanced glycosidase activity of A/J mice by a markedly change (p<0.0001) of metabolite formation rate in RGE treatment group. Noted inter-subject variability of glycosidase activity was observed among the A/J mice. While none of the mice in the Dose Response study exhibited changes in the microbiome following RGE treatment, distinct changes in microbiome composition and richness were observed after 50 mg/kg RGE treatment in the RGE Interaction study. We also identified significant changes in relative abundance of the genus Lactobacillus, which contains species that can hydrolyze RGE.III) Specific activity of enriched enzymes increased from 0.757 to 27.5 µmol/mg/min after enrichment. The overall enrichment fold and yield was 36 and 5.81%, respectively. The SDS-PAGE and LC-MS/MS analysis revealed that one unique peptide NGVLFPR (mass=801.4497, z=2) correlating to bacterial glycosdases was found. Two bacterial glycosidases (gi: 501268188 and 147736211), when overexpressed were found to hydrolyze ginsenoside Rd to F2 and C-K. [Conclusion] We have demonstrated that in vivo conversion of primary ginsenosides in RGE to the secondary and bioactive ginsenoside C-K was only mediated by microbial glycosidases. The formation of ginsenoside F2 from Rd was found, for the first time, to be the rate-limiting step in the biotransformation of Rb1 to C-K. Two bacterial glycosidases were enriched from A/J mouse feces and confirmed for the first time to hydrolyze ginsenoside Rd to F2 and C-K. Lung cancer chemoprevention of RGE is mediated by the bacterial glycosidases activites of the A/J mouse intestinal microbiota. Measurement of activity (formation of ginsenoside F2 from Rd) of such bacterial glycosidases may help differentiate potential responders of chemoprevention of RGE and non-responders, suggested by the large inter-subject variability of bacterial glycosidase activity.