Sheng Biao Wan

Methylation suppresses the proteasome -inhibitory function of green tea polyphenols

Under physiological conditions, biotransformation reactions, such as methylation, can modify green tea polyphenols (GTPs) and therefore limit their in vivo cancer‐preventive activity. Although a recent study suggested that methylated polyphenols are less cancer‐protective, the molecular basis is unknown. We previously reported that ester bond‐containing GTPs, for example (−)‐epigallocatechin‐3‐gallate [(−)‐EGCG] or (−)‐epicatechin‐3‐gallate [(−)‐ECG], potently and specifically inhibit the proteasomal chymotrypsin‐like activity. In this study, we hypothesize that methylated GTPs have decreased proteasome‐inhibitory abilities. To test this hypothesis, methylated (−)‐EGCG and (−)‐ECG analogs that can be found in vivo were synthesized and studied for their structure‐activity relationships (SARs) using a purified 20S proteasome. The addition of a single methyl group on (−)‐EGCG or (−)‐ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased. These SARs were supported by our findings from in silico docking analysis published recently. Previously, we synthesized a peracetate‐protected (−)‐EGCG molecule, Pro‐EGCG (1), to enhance its cellular permeability and stability, and current HPLC analysis confirms conversion of Pro‐EGCG (1) to (−)‐EGCG in cultured human leukemic Jurkat T cells. Furthermore, in this study, peracetate‐protected forms of methylated GTPs were added in intact Jurkat T cells to observe the intracellular effects of methylation. Peracetate‐protected, monomethylated (−)‐EGCG induced greater cellular proteasome inhibition and apoptosis than did peracetate‐protected, trimethylated (−)‐EGCG, consistent with the potencies of the parent methylated analogs against a purified 20S proteasome. Therefore, methylation on GTPs, under physiological conditions, could decrease their proteasome‐inhibitory activity, contributing to decreased cancer‐preventive effects of tea consumption. J. Cell. Physiol. 213: 252–260, 2007.

Design and Syntheses of Permethyl Ningalin B Analogues: Potent Multidrug Resistance (MDR) Reversal Agents of Cancer Cells

A series of novel N-arylalkyl-3,4-diaryl-substituted pyrrole-2,5-diones were synthesized. They exhibited promising P-gp modulating activity in a P-gp overexpressing breast cancer cell line (LCC6MDR). Compound 6 (with three methoxy groups at D-ring) displayed the highest P-gp modulating activity. 6 at 1 microM can sensitize LCC6MDR cells toward paclitaxel by 18.2-fold. Interestingly, a synergy on modulating P-gp was noted when 6 and 25 were used together (fractional inhibitory concentration index FICI = 0.42). Combination of 6 (0.5 microM) and 25 (0.5 microM) resulted in a 66-fold sensitization of LCC6MDR cells toward paclitaxel. They also reversed P-gp mediated doxorubicin (DOX) and vincristine resistance. Kinetic characterization suggests that permethyl ningalin B analogues likely act as a noncompetitive inhibitor of P-gp-mediated DOX transport (K(i) = 5.4-5.8 microM). The present study demonstrates that synthetic analogues of permethyl ningalin B can be employed as effective and safe modulators of P-gp-mediated drug resistance in cancer cells.

Sensitizing human multiple myeloma cells to the proteasome inhibitor bortezomib by novel curcumin analogs

The proteasome plays a vital role in the degradation of proteins involved in several pathways including the cell cycle, cellular proliferation and apoptosis and is a validated target in cancer treatment. Bortezomib (Velcade®, PS-341) is the first US FDA approved proteasome inhibitor anticancer drug used in the treatment of refractory multiple myeloma. In spite of its improved efficacy compared to alternative therapies, about 60% of patients do not respond to bortezomib due to the emergence of resistance. We hypothesized that novel small molecules could enhance the proteasome-inhibitory and anticancer activities of bortezomib in resistant multiple myeloma cells in vitro and in vivo. The dietary polyphenol curcumin has been shown to exert anti-cancer activity in several cancer cell lines, but the effects of curcumin in solid tumors have been modest primarily due to poor water solubility and poor bioavailability in tissues remote from the gastrointestinal tract. Here we show that the water-soluble analog of curcumin #12, but not curcumin, in combination with bortezomib could enhance the proteasome-inhibitory effect in multiple myeloma cells. Furthermore, the sensitivity of the myeloma cells to cytotoxic killing in the presence of otherwise sublethal concentrations of bortezomib was enhanced by incubation with the curcumin analog #12. These findings justify further investigation into those combinations that may yield potential therapeutic benefit.

Optimization of permethyl ningalin B analogs as P-glycoprotein inhibitors.

In the present study, a total of 9 novel permethyl ningalin B analogs have been synthesized and evaluated for their P-gp modulating activity in a P-gp overexpressed breast cancer cell line LCC6MDR. Among these derivatives, compound 12 with dimethoxy groups at rings A and B and tri-substitution at ring C with ortho-methoxyethylmorpholine, meta-bromo and para-benzyloxy groups displays the most potent P-gp modulating activity with EC50 of 423 nM to reverse paclitaxel resistance. It is non-toxic towards L929 fibroblast with IC50 greater than 100 μM and with selective index greater than 236. Its mechanism to reverse P-gp mediated drug resistance is by virtue of inhibiting transport activity of P-gp, restoring intracellular drug accumulation and eventually chemosensitizing the cancer cells to anticancer drug again. Moreover, compound 12 showed better solubility (405 ng/mL) than hit compound 1 in phosphate buffer (pH 4.0). In summary, our study demonstrates that permethyl ningalin B derivative 12 is non-toxic and efficient P-gp inhibitor that is a potential candidate to be used clinically to reverse P-gp mediated cancer drug resistance.