Dietary Supplement Vitamin C Significantly Abrogates Bortezomib-Induced Multiple Myeloma (MM) Cell Growth Inhibition
Authors: Perrone Giulia , Hideshima Teru , Ikeda Hiroshi , Okawa Yutaka , Cristea Diana , Santo Loredana , Gorgun Gullu , Calabrese Elisabetta , Raje Noopur , Baccarani Michele , Cavo Michele , Anderson Kenneth C. 
Published: October 2019

Abstract
Vitamin C (ascorbic acid, AA) has antioxydative effects and is widely taken as a food supplement. Previous studies have shown that AA inhibits bortezomib-induced cytotoxicity in human cancer cell lines in vitro thought a direct interaction of both agents. Since bortezomib is a standard treatment option in multiple myeloma (MM), we have determined whether daily oral intake of AA could alter anti-MM activities of bortezomib treatment in MM patients. We first examined plasma levels of vitamin C after oral intake of AA 1000 mg (about 15mg/kg). The mean peak plasma value was 135 μmol/l, and the median concentration at steady state in healthy volunteers ranged from 60 to 100 μmol/l. We next determined direct cytoxicity of AA in several MM cell lines, assessed by [3H]-thymidine uptake assay at 24h culture. The growth inhibition of AA at physiological concentration of 125 μM was 20% in MM1S, 15% in OPM1, 0% in RPMI8226, 17% in U266, 13% in OPM2 and 1% in H9299 cells, indicating marginal growth inhibitory effect. Higher concentration of AA (1mM), induced remarkable cytotoxicity (20–99%) in the majority of MM cell lines. To confirm inhibitory effect of AA on bortezomib-induced cell death, we treated RPMI 8226 cells with therapeutic doses of bortezomib (5nM), in the absence or presence of AA. Importantly, AA (125 μM and 250 μM) significantly inhibited bortezomib (5 ηM)-triggered growth inhibition in a dose-dependent fashion in RPMI8226 cells. We also examined the inhibition of proteasome activity by bortezomib in the presence of AA (125 μM), assessed by poly-ubiquitinated protein level in RPMI cells treated with bortezomib (20 ηM, 8h). Consistent with growth inhibition assays, anti-ubiquitin immunoblotting showed significant reduction of ubiquinated protein level in the presence of AA, confirming that AA blocked inhibition of proteasomal degradation of ubiquitineted proteins by bortezomib. Similar results were observed with other antioxidant agents (ie, LNAC). We further examined whether AA blocks other classes of proteasome inhibitors. Although AA strongly inhibited peptide boronate (MG262 and bortezomib), it did not block lactacystin or MG132-triggered proteasomal inhibition. Importantly, AA could not overcome proteasome inhibition by NPI0052, currently under evaluation in clinical trials in MM. These results confirmed that AA only blocked inhibition of proteasome activity and growth of MM cells triggered by peptide boronate proteasome bortezomib. To investigate whether a vitamin C-rich diet affects bortezomib-induced cytotoxicity we used plasma collected from healthy volunteers (n=4) taking 1000 mg AA for 4 consequtive days The mean baseline AA level was 35μmol/l, with peak concentration (120 μmol/l) at 4h after taking AA. Bortezomib treatment (10 ηM, 24h) triggered 67% growth inhibition in RPMI8226 cells, which was significantly abrogated (30%) by vitamin-C enriched plasma. We confirmed that inhibition of 20S proteasome activity by bortezomib was also markedly blocked (52% vs 7%) by those plasma samples used in growth inhibition assay. Specifically accumulation of polyubiquitinated proteins triggered by bortezomib was also reduced by the plasma. Ongoing studies in a human MM cell xenograft mouse model will delineate the inhibitory effect of AA against anti-tumor activities of bortezomib in vivo.