Title

Inhibition of Carbonic Anhydrase Activity by Green Tea antioxidant EGCG

Presentation Type

Poster

Faculty Advisor

Elena Petroff

Access Type

Event

Start Date

26-4-2023 11:00 AM

End Date

26-4-2023 12:00 PM

Description

Previous studies have shown that carbonic anhydrase (CA) and its isozymes contribute to development and progression of cancer. In several studies, an antioxidant from green tea (-)- Epigallocatechin 3-O-Gallate (EGCG) has been shown to slow down proliferation of cancer cells. We hypothesized that this effect of EGCG may be due to inhibition of CA. In addition, we tested if the products of EGCG hydrolysis, Gallic acid, and (-)-Epigallocatechin inhibit Carbonic Anhydrase. Using the electrometric Wilbur-Anderson assay, we successfully measured the CA activity and showed its inhibition by acetazolamide, a known CA inhibitor. The assay involves determination of the time T in seconds required for 4 mL of water saturated with carbon dioxide to lower pH from 8.3 to 6.3 when added to 6 mL of 0.02M Tris buffer at 0°C without (T0) and with (TE) the enzyme. The activity is defined as A = 2*(T0/TE-1) assay units (AU). In 8 blank trials, T0 was 56 ± 3 s (Mean ± SD), and with 0.001mg/10mL (~3.3nM) CA, TE = 23 ± 1 s (3084 ± 297 Wilbur-Anderson units). The same concentration of carbonic anhydrase in the presence of 10µM EGCG resulted in T = 37 ± 1 sec (1095 ± 146 Wilbur-Anderson units), p < 0.001. In the presence of 10µM Gallic Acid, T = 18.4 ± 0.4 sec (3970 ± 204 Wilbur-Anderson units), p = 0.391. In the presence of 10µM (-)-Epigallocatechin, T = 20 ± 2 sec (3606 ± 594 Wilbur- Anderson units), p=0.320. These results show that EGCG partially inhibited CA, reducing its activity by 35%. The products of EGCG hydrolysis, Gallic acid, and (-)-Epigallocatechin did not affect carbonic anhydrase activity. This supports our hypothesis that cancer protective properties of EGCG may be in part due to its inhibition of CA. Thus, EGCG and its derivatives could potentially be used for cancer therapy. This research was funded by Wehner Research Fund, MSU College of Science and Mathematics Summer Student Research Program, and the Garden State Louis Stokes Alliance for Minority Participation Program.

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Apr 26th, 11:00 AM Apr 26th, 12:00 PM

Inhibition of Carbonic Anhydrase Activity by Green Tea antioxidant EGCG

Previous studies have shown that carbonic anhydrase (CA) and its isozymes contribute to development and progression of cancer. In several studies, an antioxidant from green tea (-)- Epigallocatechin 3-O-Gallate (EGCG) has been shown to slow down proliferation of cancer cells. We hypothesized that this effect of EGCG may be due to inhibition of CA. In addition, we tested if the products of EGCG hydrolysis, Gallic acid, and (-)-Epigallocatechin inhibit Carbonic Anhydrase. Using the electrometric Wilbur-Anderson assay, we successfully measured the CA activity and showed its inhibition by acetazolamide, a known CA inhibitor. The assay involves determination of the time T in seconds required for 4 mL of water saturated with carbon dioxide to lower pH from 8.3 to 6.3 when added to 6 mL of 0.02M Tris buffer at 0°C without (T0) and with (TE) the enzyme. The activity is defined as A = 2*(T0/TE-1) assay units (AU). In 8 blank trials, T0 was 56 ± 3 s (Mean ± SD), and with 0.001mg/10mL (~3.3nM) CA, TE = 23 ± 1 s (3084 ± 297 Wilbur-Anderson units). The same concentration of carbonic anhydrase in the presence of 10µM EGCG resulted in T = 37 ± 1 sec (1095 ± 146 Wilbur-Anderson units), p < 0.001. In the presence of 10µM Gallic Acid, T = 18.4 ± 0.4 sec (3970 ± 204 Wilbur-Anderson units), p = 0.391. In the presence of 10µM (-)-Epigallocatechin, T = 20 ± 2 sec (3606 ± 594 Wilbur- Anderson units), p=0.320. These results show that EGCG partially inhibited CA, reducing its activity by 35%. The products of EGCG hydrolysis, Gallic acid, and (-)-Epigallocatechin did not affect carbonic anhydrase activity. This supports our hypothesis that cancer protective properties of EGCG may be in part due to its inhibition of CA. Thus, EGCG and its derivatives could potentially be used for cancer therapy. This research was funded by Wehner Research Fund, MSU College of Science and Mathematics Summer Student Research Program, and the Garden State Louis Stokes Alliance for Minority Participation Program.