Dietary Flavonoids Inhibit The Anti-Cancer Effects Of The Proteasome Inhibitor Bortezomib
Bortezomib has shown poor efficacy in the treatment of CLL in vivo, despite potent in vitro activity.7,8 We demonstrate that the apoptotic effect of Bortezomib on CLL can be blocked by the presence of 50% autologous plasma. This indicates the presence of survival factors or active compounds that can inhibit the killing effect of Bortezomib. This inhibitory effect is due to chemical reactions between quercetin and boronic group containing chemicals, but not related to HSA. We found that a flavonoid, quercetin, blocks Bortezomib-induced apoptosis, measured in terms of Bax conformational change and translocation, cytochrome c release, ROS generation, DYm reduction, caspase activation, externalization of phosphatidylserine (Annexin V assay) and DNA fragmentation.
Quercetin is one of the abundant flavonol-type flavonoids, commonly found in vegetables and fruits. The average daily intake of flavonoids (quercetin, myricetin, kaempferol) and two other flavone-type flavonoids (apigenin and luteolin), was estimated to be 23 mg/day, with quercetin (mean intake 16 mg/day) as the most consumed of these five flavonoids.40 Quercetin is rich in the plasma, and is extensively plasma bound, almost exclusively to human serum albumin.12 The molecular interaction between serum albumin and quercetin involves ionic interactions between the negatively charged quercetin and several ionic and polar amino acids at the putative binding site.41 Binding to albumin, quercetin elicits fluorescence. The plasma concentration of quercetin is tightly associated with its dietary intake.42 Therapeutically, it has been used in anti-aging, anti-cancer and anti-inflammatory treatment. Flavonoids, such as quercetin, myricetin, kaempferol, apigenin, also have an inhibitory effect on the chymotrypsin-like activity of 26S and 20S proteasome.15 Using computational modeling, the potential interactions of these flavonoids with the chymotrypsin site (b5 subunit) of the proteasome were detected.15 These flavonoids also have apoptosis-inducing effects on cancer cells.
Quercetin and Bortezomib share similarities with respect to proteasome inhibition and the induction of apoptosis (Table 2). They both can be used in the treatment of cancer. However, the differences between these two compounds are (1) Bortezomib is a boronic dipeptide acid and quercetin is a flavonoid; (2) Bortezomib is a ROS-inducing agent and quercetin is an anti-oxidant. The question is whether they are synergistic or counteract each other in combination. We observed that quercetin prevented Bortezomib-induced ROS generation and apoptosis in primary CLL cells. However, quercetin also blocked Bortezomib-induced apoptosis in B-cell lines in which ROS generation was not evoked. Studies from other groups demonstrated that Bortezomib-induced apoptosis in mantle cell lymphoma cells was caused by ROS generation and could be prevented by 10 mM NAC, a ROS scavenger.26 In our study, 10 mM NAC neither blocked Bortezomib-induced apoptosis in B-cell lines nor in the primary CLL cells. Quercetin induces Bax accumulation and activation at higher doses, which was associated with apoptosis, in good agreement with previous work.15 Quercetin at 20 mM concentration - which was not lethal to B-cells - blocked Bortezomib-induced Bax activation, cytochrome c release from mitochondria, caspase-3 and caspase-9 activation, and apoptosis. Moreover, neither quercetin nor Bortezomib mediated caspase-8 activation, implying that Bortezomib-induced apoptosis is via the mitochondrial/caspase-9 pathway.
Proteasome activity in human tumor samples has been measured in parallel with its activity in blood. Bortezomib-mediated proteasome inhibition in prostate and lymph node samples was similar to that in blood. Inhibition of activity in the bone marrow was approximately one half that observed in blood.43 The reasons for this differential activity include differential tissue distribution of Bortezomib, as well as differential susceptibility of cells at different tissue sites. Interestingly, these observations have not been translated to clinical efficacy in CLL, where the effects of Bortezomib used as a single agent are disappointing, but activity was seen at all sites, with a reduction in lymphocytosis, lymphadenopathy and splenomegaly.8 On the other hand, Bortezomib is highly active in myeloma, another B-lymphoid malignancy, suggesting differential sensitivity to Bortezomib according to tumor type. We found that myeloma cell lines did not show increased sensitivity to Bortezomib-induced apoptosis as compared to transformed HRC57 or lymphoma DoHH2 cells. However, while quercetin was able to inhibit Bortezomib-induced apoptosis of primary CLL, HRC57 and DoHH2 cells, this protective effect was diminished for the myeloma cell lines when they were pre-incubated with quercetin. These results suggest that quercetin (at 20 mM) has (i) a direct effect on myeloma cells, but not CLL, HRC57 or DoHH2 cells, to increase sensitivity to, and/or synergize with, Bortezomib, which is balanced by (ii) a protective effect from Bortezomib-induced apoptosis, which involves direct interaction with Bortezomib and the formation of boronate complexes. Whether dietary intake of quercetin could alter Bortezomib-mediated killing of cancer cells in patients with leukemia is unknown, but the lowest concentration tested in our study was 10 mM, which is 12.5 times higher than measured serum levels (0.8 mM) even after supplemental quercetin.24;44 Other dietary flavonoids in both catechol and pyrogallol groups are also able to inhibit Bortezomib activity in vivo, as shown for myricetin in this study and listed compounds in the Table 1. Given the ability of inorganic boric acid to restore the activity of Bortezomib in the presence of quercetin, it would be important to study the effects of both dietary quercetin, other catechols and supplemental boric acid on the susceptibility of cells to Bortezomib mediated killing in vivo and in vitro. Indeed, ingested inorganic boric acid is non toxic – it is even safert than table salt – suggesting that combined Bortezomib and boric acid may have clinical application.
Not all dietary flavonoids can inhibit Bortezomib. Myricetin showed a similar effect to quercetin on the inhibition of Bortezomib, but kaempferol and apigenin did not. Quercetin is catechol and myricetin is pyrogallol. Bortezomib is a boronic acid derivative. A notable feature of the chemistry of both boric and boronic acids is their strong tendency to form complexes with either catechols or pyrogallols.30,31 The efficacy of Bortezomib was tested in the presence of inorganic boric acid. Boric acid sensitized CLL cells to Bortezomib-induced apoptosis in a dose-dependent manner. It also abolished quercetin or serum-mediated inhibition of Bortezomib; this is consistent with competition between boric acid and Bortezomib for binding to quercetin in the plasma. Furthermore, quercetin also interferes with MG-262, another boronic acid-based proteasome inhibitor. Raman spectroscopy revealed the chemical reactions that can take place in the plasma between quercetin and Bortezomib, boric acid, or MG-262.
This study suggests that the inability of Bortezomib to induce apoptosis in circulating leukemic cells may be partly due to the presence of catechols and pyrogallols in the plasma. Quercetin did not inhibit the apoptotic activity of MG-132, a tripeptidyl aldehyde proteasome inhibitor, or lactacystin, a β-lactone-related proteasome inhibitor. Flavopiridol, a novel anti-cancer drug, is a synthetic flavone but does not contain a catechol structure in the molecule.45 It is a potent chemotherapeutic drug in the treatment of CLL46 and is also able to synergize with Bortezomib in inducing apoptosis of CLL in vitro47 – this combination could also be of interest clinically, with the lack of a catechol group meaning Flavopiridol would not exert inhibitory binding effects with Bortezomib.
In summary, quercetin, a dietary flavonoid, can block the hallmarks of Bortezomib-induced apoptosis in both cell lines and primary CLL cells. However, not all flavonoids or ROS scavengers can inhibit Bortezomib. The inhibitory effect is associated with the catechol and pyrogallol structures of some flavonoids, such as quercetin, cyaniding, myricetin, EGC, EGCG, and delphinidin, and complex formation with the boric acid group in Bortezomib. The differential in vivo activity of Bortezomib seen in myeloma and CLL may partly be attributable to the effect of dietary flavonoids: quercetin primes myeloma cells, but not CLL cells, such that they become more sensitive to Bortezomib-induced killing. Further work will elucidate the in vivo significance of these findings, which in turn will inform the need for dietary advice on the intake of flavonoids, as well as drug manipulation of flavonoid activity.
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