Ascorbic Acid Inhibits Antitumor Activity of Bortezomib in Vivo

Ascorbic acid inhibits antitumor activity of bortezomib in vivo

G Perrone1,2, T Hideshima1, H Ikeda1, Y Okawa1, E Calabrese1, G Gorgun1, L Santo1, D Cirstea1, N Raje1, D Chauhan1, M Baccarani2, M Cavo2 and KC Anderson1

1Department of Medical Oncology, LeBow Institute for Myeloma Therapeutics and Jerome Lipper Myeloma Center, Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA, USA and 2Department of Hematology, Sera`gnoli Institute of Hematology and Medical Oncology, Bologna University School of Medicine, Bologna, Italy

Earlier studies have shown that ascorbic acid (vitamin C) the tissue.8,9 Although epidemiological studies support a role for inhibits bortezomib-induced cytotoxicity against cancer cells in vitamin C in preventing cancer,10 its influence on both tumor vitro. However, the clinical significance of vitamin C on bortezomib treatment is unclear. In this study, we examined and activity of chemotherapeutic agents remains controversial. whether daily oral intake of vitamin C inhibits antimultiple Several preclinical studies suggest that vitamin C can increased 11,12 myeloma (MM) activities of bortezomib. Vitamin C, at orally the efficacy of cancer agents; however, more recent studies achievable concentrations, inhibited in vitro MM cell cytotoxi- have shown that dehydroascorbic acid, the oxidant form of city of bortezomib and blocked its inhibitory effect on 20S vitamin C, can antagonize the effect of conventional antineo- proteasome activity. Specifically, plasma collected from healthy plastic agents by preserving the stability of mitochondrial volunteers taking 1 g/day vitamin C reduced bortezomib- 13 induced MM cell death in vitro. This antagonistic effect of membrane potential in cancer cells. vitamin C against proteasome inhibitors is limited to the Bortezomib is the first proteasome inhibitor approved by the boronate class of inhibitors (bortezomib and MG262). In vivo Food and Drug Administration for the treatment of relapsed activity of this combination treatment was then evaluated using refractory, relapsed and newly diagnosed multiple myeloma our xenograft model of human MM in SCID (severe combined (MM).14–16 Moreover, combination therapies of bortezomib immune-deficient) mice. Bortezomib (0.1 mg/kg twice a week for with doxil17,18 are approved by the Food and Drug Administra- 4 weeks) significantly inhibits in vivo MM cell growth, which tion, and with heat-shock protein 90,19 AKT20 and histone was blocked by oral vitamin C (40 mg/kg/day). Therefore, our 21–24 results for the first time show that vitamin C can significantly deacetylase (HDAC) inhibitors are being evaluated in reduce the activity of bortezomib treatment in vivo; and phase III trials. Despite the encouraging results observed with importantly, suggest that patients receiving treatment with the proteasome inhibitor alone or in combination, resistance bortezomib should avoid taking vitamin C dietary supplements. develops in most cases. Importantly, earlier studies have shown Leukemia (2009) 23, 1679–1686; doi:10.1038/leu.2009.83; that vitamin C blocks the activity of bortezomib in vitro by its published online 16 April 2009 direct binding to bortezomib.25–27 In addition, production of Keywords: multiple myeloma; ascorbic acid; antioxidant; 28 bortezomib reactive oxygen species by bortezomib is reduced by vitamin C.29 However, the clinical significance of vitamin C intake in MM patients receiving bortezomib treatment remains unclear. Our study confirms that vitamin C as a dietary supplement can inhibit anti-MM activity of bortezomib in vivo. Introduction

The increasing use of antioxidative dietary supplements in Materials and methods cancer patients has renewed concerns about their potential impact on tumor cells and therapies.1 Besides their ability to Cell culture reduce treatment-related side effects2 and increase the sensitiv- MM1S cells were kindly provided by Dr Steven Rosen (North- ity of tumor cells to chemotherapeutic agents,3 antioxidative western University, Chicago, IL, USA); OPM1 cells by Dr P Leif agents can also decrease the efficacy of treatments by reducing Bersagel (Mayo Clinic, Scottsdale, AZ, USA); and OPM2 cells by their induction of oxidative stress.4 However, to date, experi- Dr Edward Thompson (University of Texas Medical Branch, mental data and clinical studies have not definitively shown Galveston, TX, USA). RPMI8226, U266 and H929 cells were impact on patient’s outcome.1,5,6 obtained from American Type Culture Collection (Rockville, Vitamin C (ascorbic acid), a potent water-soluble antioxidant MD, USA). MM cell lines were cultured in RPMI1640 medium in plant and animal cells, is among the most common dietary supplemented with 10% heat-inactivated fetal bovine serum m supplement in cancer patients. The median level of vitamin C in (Sigma Chemical Co., St Louis, MO, USA), 2 ML-glutamine and the plasma after oral ingestion ranges between 60–100 mmol/l, 100 U/ml penicillin (Life technologies, Inc., Grand Island, NY, but higher levels (220 mmol/l) are achievable.7 Moreover, USA). vitamin C can accumulate as ascorbic acid, reaching millimolar intracellular concentrations depending on the characteristics of Vitamin C and proteasome inhibitors Vitamin C and L-acetyl cysteine were purchased from Correspondence: Professor KC Anderson, Department of Medical Sigma-Aldrich (St Louis, MO, USA). Vitamin C was dissolved Oncology, Dana-Farber Cancer Institute, Mayer 557, 44 Binney Street, in sterile distilled water and prepared immediately before use. Boston, MA 02115, USA. E-mail: [email protected] Vitamin C for human study was purchased from commercial Received 3 February 2009; revised 13 March 2009; accepted 18 sources (Vitamin C, tablet 1000 mg, from CVS Pharmacy Inc., March 2009; published online 16 April 2009 Woonsocket, RI, USA). Bortezomib was purchased from Vitamin C supplement in multiple myeloma G Perrone et al 1680 Millennium Pharmaceuticals Inc. (Cambridge, MA, USA). Xenograft murine model of human MM MG132, MG262 and lactacystin were obtained from Calbiochem Fox-chase SCID (severe combined immune-deficient) mice (EMD Biosciences, San Diego, CA, USA). NPI-0052 was obtained (18–20 weeks old) were purchased from Charles River from Nereus Pharmaceuticals (San Diego, CA, USA). Laboratories (Wilmington, MA, USA). All animal studies were conducted according to protocols approved by the Animal Ethics Committee of the Dana-Farber Cancer Institute. DNA synthesis An initial pharmacokinetic study to evaluate the peak level of To assess the growth of MM cells, the rate of DNA synthesis was vitamin C was carried out in a cohort of six mice. Vitamin C measured as described earlier.30 Briefly, MM cells (3 105 cells (40 mg/kg) was given orally for 4 days, and blood samples were per well) were incubated in 96-well culture plates in the collected 2 or 4 h after the last intake for measurement of presence of vitamin C or of plasma from healthy donors for 24 or vitamin C plasma levels. We next examined the effect of vitamin C on bortezomib treatment in vivo. Mice were irradiated 48 h, with or without proteasome inhibitors. Cells were pulsed 6 3 3 with 0.5 uCi/well of [ H] thymidine ([ H]-TdR; Perkin Elmer, (200 rad), and then 5 10 RPMI8226 cells were inoculated Boston, MA, USA) during the last 6 h of culture, harvested onto subcutaneously in the right flank. When tumor was measurable, glass filters with an automatic cell harvester (Cambridge mice were assigned into three cohorts (four mice each) receiving Technology, Cambridge, MA, USA), and counted using an vehicle i.v. saline solution (0.9% NaCl following the schedule of LBK Betaplate scintillation counter (Wallac, Gaithersburg, MD, bortezomib), bortezomib i.v. (0.1 mg/kg on days 1, 4, 8, 11, 14, USA). 17, 22 and 26), or bortezomib plus vitamin C (40 mg/kg/day). Caliper measurement of the longest perpendicular tumor diameters was carried out every alternate day to estimate the tumor volume, using the following formula: 4p/3 (width/2)2 Immunoblotting (length/2), representing the three-dimensional volume of Multiple myeloma cells were harvested and lysed using radio- an ellipse. Mice were killed at the end of the first cycle of immunoprecipitation assay lysis buffer (5 mM EDTA (ethylene- treatment, 1 h after the 8th injection, or when tumor reached diaminetetraacetic acid), 2 Mm Na3VO4, 5 mM NaF and 1 mM 2cm3; plasma and tumor from mice were immediately collected PMSF (phenylmethanesulphonylfluoride or phenylmethylsul- and evaluated. phonyl fluoride)) as described before.7 The whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (BIO-RAD Laboratories, Hercules, CA, USA) and immunoblotted with antiubiquitin (Cell Statistical analysis Signaling, Beverly, MA, USA), a-tubulin or antiactin antibodies Statistical significance of differences in tumor growth observed (Santa Cruz Biotech, Santa Cruz, CA, USA). between treated and untreated groups was determined using unpaired Student’s two-tailed t-tests; results are presented as mean±s.e. Statistical significance of differences in proteasome activity between bortezomib versus vitamin C plus 20S proteasome inhibitor assay bortezomib treatment was determined using two-way analysis Cell lysates from RPMI8226 cells were assessed for 20S of variance test; all statistical analyses were carried out using proteasome activity, based on the rate of the chymotryptic Graph Pad Prism software (Graph Pad Software Inc., San Diego, subunit cleavage of a fluorescent molecule 7-amino-4-methyl- CA, USA). coumarin (AMC) labeled pentapeptide, as described earlier.31

Results Quantification of vitamin C Vitamin C levels in the plasma from healthy donors and mice Vitamin C blocks bortezomib-induced growth inhibition were measured using a colorimetric assay kit, according to the in vitro manufacturer’s instruction (Ferric Reducing Ascorbate Assay Kit, We first determined the effect of vitamin C on growth of MM cell BioVision, CA, USA). lines. MM1S, OPM1, RPMI8226, U266, OPM2 and H929 MM cells were cultured with increasing doses (30–500 mM)of vitamin C for 24 h. As shown in Figure 1a, the growth of MM Vitamin C-rich plasma culture cell lines was minimally inhibited by vitamin C, even at high An initial study to assess pharmacokinetics of ascorbic acid was micromolar concentrations. We next examined whether vitamin carried out in healthy volunteers receiving 1 g/day of vitamin C C inhibits bortezomib-induced growth inhibition in vitro. orally for 4 consecutive days (15–20 mg/kg/day). Peripheral RPMI8226 cells were cultured with bortezomib (5–10 nM)in blood was collected on the 4th day at 0, 4 and 6 h post- the presence (30–500 mM) or absence of vitamin C. Consistent treatment under the auspices of an institutional review board- with earlier studies,25,27 vitamin C almost completely blocked approved protocol. Plasma was separated, and vitamin C level bortezomib-induced growth inhibition in a dose-dependent was measured as described above. In proliferation assays, fashion (Figure 1b). For example, 7.5 nM of bortezomib induced 3 RPMI8226 cells were cultured for 24 h in 96-well plates with 95% inhibition of [ H] thymidine uptake, whereas 125 mM plasma, in the presence or absence of bortezomib. vitamin C reduced its inhibition to 30%. To determine whether Subsequently, we carried out a dose-escalation study in the antagonistic effect of vitamin C against bortezomib healthy volunteers receiving vitamin C supplements for 7 days. treatment was associated with a block in proteasome inhibition, The dose of vitamin C was increased every other day from 250 we next examined polyubiquitinated protein levels and 20S to 500 and 1000 mg. Blood samples were collected at baseline proteasome activity. Bortezomib significantly increased the and 4 h after the last vitamin C intake. Plasma was separated, levels of polyubiquitinated protein, which was blocked in the and vitamin C level was measured as described above. presence of vitamin C (125 mM; Figure 1c). Moreover, vitamin C

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Figure 1 Vitamin C blocks the activity of bortezomib in vitro.(a) MM1S (K), OPM1 (m), RPMI8226 (&), U266 (J), OPM2 (’) and H929 (n). 3 MM cells were cultured with increasing doses (range: 30–500 mM) of vitamin C. After 24 h culture, growth inhibition was assessed by [ H] thymidine uptake. Values represent the mean (±s.d.) of triplicate cultures relative to control culture in media alone. (b) RPMI8226 cells were cultured with different doses of bortezomib (range: 5–10 nM) in the presence ( 30 mM, 61.5 mM, 125 mM, 250 mM, 500 mM) or absence of vitamin C. [3H] thymidine uptake was assessed at 24 h. Values represent the mean (±s.d.) of triplicate cultures relative to control cultures in media alone. (c) RPMI8226, MM1S and OPM1 cells were cultured in the presence of vitamin C (125 mM) for 24 h, with or without bortezomib (5 nM). Cells lysates were subjected to immunoblotting to assess polyubiquitinated protein levels. Antiactin immunoblotting was used as a loading control. (d) 20S proteasome activity was assessed in RPMI8226 cells treated with bortezomib (5 nM) for 24 h, in the presence or absence of vitamin C (125 mM). The results are expressed as percentage of protease activity compared with control in media alone.

signiﬁcantly (Po0.05) blocked the inhibition of the 20S used as an antioxidant, blocked the growth inhibitory effect of proteasome activity triggered by bortezomib (Figure 1d). all of these proteasome inhibitors (Figure 2c). These results indicate that vitamin C selectively blocks peptide boronate class of proteasome inhibitors, and that oxidative stress plays a major Vitamin C blocks peptide boronate class proteasome role in proteasome inhibitor-induced growth inhibition in MM. inhibitors To test the hypothesis that the inhibition of the activity of bortezomib is due to a direct binding between vitamin C and Plasma from healthy donors taking vitamin C dietary boronic acid, we next examined whether vitamin C could also supplements inhibits bortezomib activity inhibit other classes of proteasome inhibitors. Although vitamin We next examined the relevance of vitamin C plasma levels C markedly blocked MG262 (peptide boronate) and bortezo- achieved after dietary supplements on bortezomib-induced MM mib-induced growth inhibition in a dose-dependent fashion cell growth inhibition. A peak concentration of 150 mM vitamin (Figure 2a), it did not abrogate the effect of NPI0052 (data not C was observed 4 h after oral intake of 1 g vitamin C tablet, shown), lactacystin or MG132 (peptide aldehyde; Figure 2b). which decreased to baseline levels after 6 h (Figure 3a). On the other hand, L-acetyl cysteine (LNAC), most commonly Although vitamin C-enriched plasma alone did not trigger

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Figure 2 Vitamin C inhibits only boronate proteasome inhibitors. (a) RPMI 8226 cells were cultured with MG 262 (5 nM) or bortezomib (5 nM), 3 with or without sequential doses of vitamin C ( 125 mM and 250 mM). [ H] thymidine uptake was assessed after 24 h relative to control cultures in media alone. (b) RPMI8226 cells were cultured with bortezomib (7.5 and 10 nM), lactacystin (5 and 10 mM) or MG132 (0.25 and 0.5 mM), in the 3 absence or presence of vitamin C ( 250 mM and 500 mM). [ H] thymidine uptake was assessed after 24 h relative to media alone cultures. (c) RPMI8226 cells were cultured with bortezomib (7.5 and 10 ZM), lactacystin (5 and 10 mM) or MG132 (0.25 and 0.5 mM), in the absence or 3 presence of L-NAC ( 0.5 and 1mM). [ H] thymidine uptake was assessed after 24 h relative to media alone cultures. L-Nac, L-acetyl cysteine

cytotoxicity, it markedly reduced bortezomib-induced growth doses of vitamin C (31, 62, 125, 250 500 mM) and bortezomib inhibition. For example, bortezomib alone triggered 76% (10 nM). As shown in Figure 3c, the reduction of bortezomib growth inhibition; however, plasma at 2 and 4 h after vitamin induced-cytotoxicity invereses linearly with the dose of vitamin C intake decreased its inhibition to 56% (Figure 3a). This result C and reaches a plateau at levels higher than 125 mM of vitamin suggests that vitamin C as a dietary supplement blocks C. Our results suggest that even levels of ascorbic acid present at bortezomib-induced cytotoxicity in MM cells in vitro. baseline in healthy volunteers (range:38–79 umol/l) can reduce We next evaluated the impact of lower doses of vitamin C bortezomib-induced cytotoxicity (90 versus 63% of growth supplements to assess whether there is a dose that does not inhibition, with 0 and 62 mM of vitamin C, respectively). More- affect bortezomib-induced cytotoxicity. Plasma samples were over, 125 mM of vitamin C levels, attained 4 h after ingestion of collected from healthy volunteers taking increasing doses of 500 mg of vitamin C supplements, strongly inhibits bortezomib vitamin C supplements (250, 500 and 1000 mg). The average cytotoxicity (90 versus 22% of growth inhibition, with 0 and vitamin C plasma level measured was 57 umol/l (range: 125 mM of vitamin C). 38–79 umol/l), 75 umol/l (range: 64–82 umol/l), 101 umol/l (range:84–115 umol/l) and 115 umol/l (range: 96–153 umol/l) at baseline and 4 h after ingestion of 250, 500 and 100 mg of Vitamin C abrogates in vivo anti-MM activity of vitamin C (Figure 3b). Subsequently, RMPI8226 cells were bortezomib cultured in 10% plasma collected from healthy volunteers who To further investigate the clinical relevance of the antagonistic are not taking vitamin C supplements in the presence of different effect of vitamin C against bortezomib, we next carried out

Leukemia Vitamin C supplement in multiple myeloma G Perrone et al 1683

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Figure 3 Plasma rich in vitamin C reduces the activity of bortezomib. (a) Plasma was collected from healthy donors at different time points (0, 2, 4 and 6 h) after oral intake of 1 g vitamin C . RPMI8226 cells were cultured in the presence of this plasma with (’) or without (&) bortezomib 3 (20 ZM). [ H] thymidine uptake was assessed after 24 h of culture relative to control. The continuous line represents the vitamin C plasma level (m). Three separate experiments were conducted in triplicate; results shown represent the mean (SD±50) of a representative experiment. (b) Vitamin C plasma level was measured in healthy voluntaries at baseline and 4 h after different doses of vitamin C oral intake (250, 500 and 1000 mg). (c) RMPI8226 cells were cultured in 10% human plasma with different doses of vitamin C (31, 62, 125, 250 and 500 mM), with or without 3 bortezomib (10 ZM). [ H] thymidine uptake was assessed after 24 h of culture relative to control. in vivo studies of this combination in our xenograft mouse tumor growth as the control group (K; Figure 4b). For example, model of human MM. Mice were treated with vitamin C after the first cycle of therapy, mice treated with bortezomib (40 mg/kg/day) orally, approximately 0.04% of their average alone had a significantly lower tumor volume compared diet, which does not affect intracellular levels of vitamin C and with mice receiving bortezomib plus vitamin C supplement results in only transient fluctuations in blood concentrations.32 (137±39.12 versus 507.5±100.6 mm3; P ¼ 0.014), indicating The plasma level of vitamin C peaked 2 h after vitamin C intake that vitamin C antagonized anti-MM activity of bortezomib (Figure 4a), suggesting that kinetics of vitamin C in mice is in vivo. Tumor tissues were collected to confirm the different than in man (Figure 3a). block of proteasome inhibition in the bortezomib plus vitamin Mice were then treated with bortezomib, as described earlier. C-treated cohort. As shown in Figure 4c, the increase in As vitamin C alone did not appreciably alter tumor growth, we ubiquitinated proteins induced by bortezomib alone was compared cohorts of mice treated with vehicle control (K), completely blocked when vitamin C was added to bortezomib. bortezomib (’) and bortezomib plus vitamin C (m). Bortezomib These data confirm that vitamin C as a dietary supplement significantly inhibited tumor growth (P ¼ 0.0004); how- can significantly reduce the efficacy of bortezomib treatment ever, vitamin C plus bortezomib treatment showed similar in vivo.

Leukemia Vitamin C supplement in multiple myeloma G Perrone et al 1684 a 100

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Figure 4 Vitamin C attenuates the cytotoxicity of Velcade treatment in vivo.(a) Vitamin C plasma level was measured in SCID mice before, 2 and 4 h after vitamin C (40 mg/kg) oral intake. (b) SCID mice were injected subcutaneously with RPPMI8226 cells and treated for 4 weeks with vehicle control (K), bortezomib 0.1 mg/kg (’) on days 1, 4, 8, 11, 14, 17, 22 and 26, or bortezomib 0.1 mg/kg plus VITAMIN C 40 mg/kg/daily (m). Tumor volume was measured; error bars represent mean±s.e. (c) Tumor was harvested after the last injection. Whole tumor lysates were immunoblotted with antiubiquitin antibody to assess proteasome activity. Anti-a tubulin was used as a loading control. SCID, severe combined immune-deﬁcient.

Discussion of the proteasome. In this study, we show that vitamin C at physiological concentration blocks bortezomib both in vitro Recent studies have shown that vitamin C can inhibit the in vitro and in vivo. activity of bortezomib through a direct binding between the After confirming the low toxic profile of vitamin C alone on hydroxyl group of the antioxidant agent and the boronic acid MM cell lines, we showed that the inhibition on bortezomib of the proteasome inhibitor,25–27 thereby reducing the affinity by vitamin C is dose dependent. We also showed that this of the proteasome inhibitor for the chymotrypsin-like subunit inhibitory effect of vitamin C was restricted to proteasome

Leukemia Vitamin C supplement in multiple myeloma G Perrone et al 1685 inhibitors containing a peptide boronic acid, consistent with 5 Hoffer LJ, Levine M, Assouline S, Melnychuk D, Padayatty SJ, earlier studies in cancer cell lines.25–27 In contrast, L-acetyl Rosadiuk K et al. Phase I clinical trial of i.v. ascorbic acid in cysteine, another common antioxidant dietary supplement, advanced malignancy. Ann Oncol 2008; 19: 1969–1974. inhibits cell death induced by proteasome inhibitors regardless 6 Pathak AK, Bhutani M, Guleria R, Bal S, Mohan A, Mohanti BK 33 et al. Chemotherapy alone vs chemotherapy plus high dose of their chemical structure. These data should be carefully multiple antioxidants in patients with advanced non-small cell interpreted, as the uptake of ascorbic acid is less efficient and lung cancer. J Am Coll Nutr 2005; 24: 16–21. reproducible in vitro than in vivo.34 Nevertheless, these data 7 Padayatty SJ. Vitamin C pharmacokinetics: implication for oral and suggest that the activity of bortezomib can be attenuated either intravenous use. Ann Int Med 2004; 140: 533–538. by direct blocking by Vitamin C or by inhibition of the 8 Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH et al. downstream pathway by L-acetyl cysteine. Vitamin C as an antioxidant: evaluation of its role in disease prevention. J Am Coll Nutr 2003; 22: 18–35. Most importantly, plasma collected from donors receiving a 9 Levine M, Conry-Cantilena C, Wang Y, Welch RW, Washko PW, daily supplement of vitamin C reduced the activity of Dhariwal KR et al. Vitamin C pharmacokinetics in healthy bortezomib level in vitro, both its proteasome inhibitor activity volunteers: evidence for a recommended dietary allowance. and cytotoxicity. Our data suggest that even a baseline value of Proc Natl Acad Sci USA 1996; 93: 3704–3709. vitamin C present in healthy donors can inhibit the activity of 10 Head KA. Ascorbic acid in the prevention and treatment of cancer. bortezomib. Moreover, our in vitro findings were confirmed Altern Med Rev 1998; 3: 174–186. 11 Nagy B, Mucsi I, Molnar J, Varga A, Thurzo L. Chemosensitizing in vivo using our xenograft mouse model of MM: the efficacy of effect of vitamin C in combination with 5-fluorouracil in vitro. Bortezomib in inhibiting human MM cell growth and prolonging In Vivo 2003; 17: 289–292. host survival were reduced when mice received bortezomib 12 Kurbacher CM, Wagner U, Kolster B, Andreotti PE, Krebs D, with vitamin C supplements. Although the precise mechanism Bruckner HW. Ascorbic acid (vitamin C) improves the antineo- and the kinetics of vitamin C binding to bortezomib in vivo plastic activity of doxorubicin, cisplatin, and paclitaxel in human is not completely known, our data nevertheless confirms that breast carcinoma cells in vitro. Cancer Lett 1996; 103: 183–189. clinically relevant levels of vitamin C block bortezomib-induced 13 Heaney ML, Gardner JR, Karasavvas N, Golde DW, Scheinberg DA, Smith EA et al. Vitamin C antagonizes the cytotoxic effects cytotoxicity. We, therefore, suggest that vitamin C supplements of antineoplastic drugs. Cancer Res 2008; 68: 8031–8038. should be avoided in patients receiving proteasome inhibitor 14 Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, therapy. In particular, we recommend that patients who are Irwin D et al. A phase 2 study of bortezomib in relapsed, refractory under treatment with bortezomib should not take more than myeloma. N Engl J Med 2003; 348: 2609–2617. 500 mg of vitamin C. Specifically, it should be avoided at least 15 Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer 12 h before and after bortezomib treatment as the half-life of oral EA, Facon T et al. Bortezomib or high-dose dexamethasone for 7,35 relapsed multiple myeloma. N Engl J Med 2005; 352: 2487–2498. ascorbic acid in plasma is 10 h. 16 San Miguel JF, Schlag R, Khuageva NK, Dimopoulos MA, The transport and the accumulation of vitamin C depend on Shpilberg O, Kropff M et al. Bortezomib plus melphalan and several mechanisms, including diet as well as modulating prednisone for initial treatment of multiple myeloma. N Engl J Med factors, such as proton pump inhibitors,36 which increase gastric 2008; 359: 906–917. pH and reduce the stability and absorption of vitamin C; or 17 Orlowski RZ, Nagler A, Sonneveld P, Blade J, Hajek R, Spencer A dexamethasone, which induces sodium dependent vitamin C et al. Randomized phase III study of pegylated liposomal transporters and increases the accumulation of ascorbic acid, doxorubicin plus bortezomib compared with bortezomib alone 37 in relapsed or refractory multiple myeloma: combination therapy as shown in an osteoclastic cell model. Importantly, our study improves time to progression. J Clin Oncol 2007; 25: 3892–3901. shows that vitamin C supplementation decreases the efficacy of 18 Sonneveld P, Hajek R, Nagler A, Spencer A, Blade J, Robak T et al. bortezomib treatment and outcome in vivo even at relatively Combined pegylated liposomal doxorubicin and bortezomib is low concentrations. Besides vitamin C, other natural agents highly effective in patients with recurrent or refractory multiple carrying a hydroxyl groups, such as flavonoid compounds as myeloma who received prior thalidomide/lenalidomide therapy. quercetin,2 bind and inhibit the activity of bortezomib in vitro. Cancer 2008; 112: 1529–1537. 19 Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Kung AL, Taken together, these studies suggest that antioxidant supple- Davies FE et al. Antimyeloma activity of heat shock protein-90 ments should be avoided in patients receiving boronic acid inhibition. 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