Resveratrol given intraperitoneally does not inhibit the growth of high-risk t(4;11) acute lymphoblastic leukemia cells in a NOD/SCID mouse model

Abstract

The efficacy of resveratrol as a preventive agent against the growth of t(4;11) acute lymphoblastic leukemia (ALL) was evaluated in NOD.CB17-Prkdcscid/J mice engrafted with the human t(4;11) ALL SEM cell line. SEM cells were injected into the tail vein and engraftment was monitored by flow cytometry. Once engraftment was observed, mice were injected intraperitoneally with resveratrol (10 mg/kg body weight) dissolved in dimethylsulfoxide (DMSO) or DMSO alone (control) every other day, or vincristine (0.5 mg/kg body weight) 3 times per week for 4 weeks (n=16 per group). Comparisons of the percent of human leukemia cells in blood and survival curves showed resveratrol did not inhibit progression of the disease. Liquid chromatography-tandem mass spectrometry analyses of mouse sera showed resveratrol was rapidly metabolized to glucuronidated and sulfated forms 1 h post-injection, with low to no resveratrol or metabolites observed in sera by 24-48 h. These data indicate that in contrast to findings in in vitro models, parenterally administered resveratrol does not have potential as a preventive agent against high risk t(4;11) ALL.

Figure 1

Resveratrol at a concentration of 40 mg/kg body weight was toxic to NOD/SCID mice engrafted with t(4;11) acute lymphoblastic leukemia (ALL). Mice were engrafted with the t(4;11) ALL line SEM, and then treated intraperitoneally with either DMSO (n=13) or resveratrol (40 mg/kg body weight, n=14) daily, or vincristine (0.5 mg/kg body weight, n=13) one time per week for 4 weeks. Mice were euthanized when they became clinically ill, i.e., showed >20% weight loss, lethargy, weakness, or inability to reach food or water. Differences in survival after treatment began were determined by log-rank test. The asterisk indicates a significant reduction in survival for resveratrol-treated mice compared to the DMSO and vincristine treated mice (p<0.05).

Figure 2

Resveratrol at a concentration of 10 mg/kg body weight did not increase survival of leukemic mice. Three groups of mice were engrafted with the t(4;11) ALL line SEM, and then treated intraperitoneally with either DMSO or resveratrol (10 mg/kg body weight) every other day, or vincristine (0.5 mg/kg body weight) three times per week for 4 weeks (n=16 per treatment group). Mice were euthanized when they became clinically ill as described in Fig. 1. Differences in survival after treatment began were determined by log-rank test. The asterisk indicates a significant difference in survival between vincristine and DMSO or resveratrol treated mice (p<0.05).

Figure 3

Loss of body weight was associated with increasing leukemia burden. Three groups of mice were engrafted with the t(4;11) ALL line SEM, and then treated intraperitoneally with either DMSO or resveratrol (10 mg/kg body weight) every other day, or vincristine (0.5 mg/kg body weight) three times per week for 4 weeks (n=16 per treatment group). (A) Body weight for each mouse was measured weekly beginning at age of 6 weeks. The asterisks indicate a difference in body weight for vincristine-treated mice compared to DMSO and resveratrol-treated mice (p<0.05). The arrow denotes the age of mice when the leukemia cells were injected. (B) PBLs were isolated from each mouse and stained with PE-Cy7 conjugated anti-human CD19 and APC-Cy7 conjugated anti-mouse CD45. The proportion of human CD19⁺ cells in the murine PBL population was monitored weekly by flow cytometry beginning 2 weeks after the injection of leukemia cells (age of 10 weeks) until the treatment period ended. The asterisks indicate a difference in the percent of CD19⁺ in the DMSO and resveratrol-treated mice compared to vincristine treated mice (p<0.05). Data represent means ± SD.

Figure 4

Engraftment sites for the SEM t(4;11) leukemia cells. Mice were engrafted with the t(4;11) ALL line SEM and treated with DMSO every other day for 4 weeks. Blood, spleens, and bone marrow were collected from 4 mice in the DMSO control group following euthanasia. Splenocytes were prepared by first perfusing with tissue with medium, and shredding to release the cells. The red blood cells were lysed in the blood and splenocyte preparations with PharmLyse. Both femurs from each mouse were removed into medium and the bone marrow was removed by perfusing the inside of the bone with medium. PBLs, splenocytes, and bone marrow cells were stained with PE-Cy7 conjugated anti-human CD19 and APC-Cy7 conjugated anti-mouse CD45 and analyzed by flow cytometry. Data are representative of four mice.

Figure 5

Leukemic NOD/SCID mice retain the ability to metabolize resveratrol. (A) Representative UPLC-MS/MS chromatograms of resveratrol isolated from serum collected 1 h post resveratrol i.p. injection after digestion with buffer (i.e., mock), sulfatase, or β-glucuronidase. Deconjugation reactions increased peak areas of resveratrol in serum relative to mock digestions demonstrating the rapid generation of glucuronide and sulfate conjugates in vivo. (B) At 1 h post-injection of resveratrol, mean plasma concentrations of total resveratrol metabolites were estimated at ~4±2 μM from 5 animals, with 17/58/25% distribution between parent, glucuronidated and sulfated metabolites. Data were presented as percent ± SEM. Concentrations were below the level of accurate quantification within 24 h, and the frequency of target analyte detection reduced from 67 to 30% between 24 and 48 h post-injection. LOD, limit of detection.