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Supplemental Figures and Tables
Supplemental Figure 1. Elevated NQO1 expression in patients with HNC
correlates with poor overall survival to standard therapy. Overall survival
associated with NQO1 gene expression was assessed in 286 head and neck cancer
patients using the PROGgeneV2 online webtool with data sourced from The Cancer
Genome
Atlas
(TCGA)
through
the
following
URL:
(http://watson.compbio.iupui.edu/chirayu/proggene/database/?url=proggene) [1]. Patient
cohorts were divided into “High” (145 patients) and “Low” (141 patients) NQO1 gene
expression groups based on median gene expression. The raw data from this analysis
was plotted as a Kaplan-Meier overall survival plot in GraphPad Prism and statistical
significance was calculated using the log-rank test. Overall survival data indicated that
patients with high NQO1 expressing tumors have significantly reduced survival
compared to low expressing HNC cancers (p = 1 X 10-4).
Supplemental Figure 2. Functional shRNA-NQO1 knockdown rescues β-lapinduced cell death. (A) Lentiviral shRNA against NQO1 was used to stably reduce the
protein abundance of NQO1 in FaDu cells and a representative western blot for several
different independent NQO1 knockdowns is presented. A non-targeting scrambled
shRNA (shSCR) was used to control for the lentiviral vector and GAPDH served as the
loading control. (B) β-Lap-induced lethality is rescued in FaDu shNQO1 knockdown
clones. Relative survival of shSCR and shRNA-NQO1 knockdown cells #6 and #7 were
evaluated after various doses of β-lap exposure (µM, 2 h) as depicted on the x-axis
demonstrating a β-lap induced NQO1 dependent cell death (***p < 0.001).
Supplemental Figure 3. β-Lap-induced NQO1 dependent DNA damage and NAD+
depletion. Time-dependent increase in DNA strand breaks in FaDU cells was
demonstrated by the alkaline comet assay in the presence of β-lap alone (5 µM, 2 h) or
co-treatment with β-lap and DIC (50 μM). (A) Shown are representative micrographs of
studies performed at least three times and (B) the graphed comet tail lengths were
measured using Komet 5.5 software showing means ± SE from three experiments.
Li et al
Student’s t tests were performed to assess significance (***p < 0.001). (C) NAD+ levels
are dramatically reduced by exposure to β-lap at the indicated doses and this response
is blocked by co-treatment with DIC (50 μM) in FaDu and SqCC/Y1 cells. Student’s t
tests were performed to assess for significance (***p < 0.001).
Supplemental Figure 4. Combination sublethal doses of β-lap and low dose IR
induce NQO1 dependent anti-cancer lethality. Relative cell survival assays were
performed by treating with sublethal doses of β-lap at the specified concentration (M 2
h) immediately after 2 Gy exposure, with or without co-treatment of DIC (50 M) in
SqCC/Y1 cells. The triple asterisks represent statistically significant differences between
the β-lap (2.5 M) + 2 Gy vs. β-lap (2.5 M) + 2 Gy + DIC (50 M) treatments (***p <
0.001). A lethal dose of β-lap (6 M), with or without DIC (50 M) served as controls.
Supplemental Figure 5. Cooperative antitumor efficacy using a combination of IR
and β-lap to treat HNC xenograft models. Mice bearing 30 mm3 SqCC/Y1 HNC
xenografts with high levels of NQO1 expression (A and B are SCID-NOD data with 10
mice for each treatment group; C and D are Athymic nu/nu phenotype with 5 mice for
each treatment group) were treated with 2 or 4 Gy every other day for five treatments
(10 and 20 Gy total, respectively). β-Lap-HPβ-CD 10 mg/kg was intravenously injected
by tail-vein immediately following IR treatment. Vehicle alone (HPβ-CD) served as a
control cohort. Results (means ±SE) are representative of repeated similar experiments
(n=10 for each group). Student’s t-tests (*** p < 0.001) were performed comparing
treated vs control groups. (A and C) Tumor volume measurements and (B and D)
Kaplan-Meier overall survival over the indicated number of days is graphed for control
(HPβ-CD), β-lap-HPβ-CD 10 or 20 mg/kg alone, 2 or 4 Gy alone and a combination of 2
or 4 Gy plus β-lap-HPβ-CD 10 mg/kg for the NOD-SCID cohorts. The nu/nu cohorts did
not receive β-lap-HPβ-CD 20 mg/kg alone, 4 Gy IR alone, or any combination that
utilized these doses. Log-rank analyses were performed comparing survival curves
using various β-lap-HPβ-CD + IR regimens (*** p < 0.001 for the combined treatment
compared to each single treatment).
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Li et al
Figure 6. Expression of Ki-67 after combination of β-lap and IR in HNC xenograft
models. Ki-67 (Santa Cruz) IHC staining was performed on 5 μm thick SqCC/Y1 HNC
formalin fixed paraffin embedded slides via two-step assays as previously described
(22). Cytoplasmic and nuclear staining was independently evaluated by two
Pathologists (Drs. Lin and Liu) in a blinded manner. Shown are nuclear staining of Ki-67
in the control (HPβ-CD), β-lap-HPβ-CD 10 mg/kg alone, 2 Gy alone and a combination
of 2 Gy plus β-lap-HPβ-CD 10 mg/kg.
Supplemental Table 1. NQO1 enzyme activity and abrogation of activity in 41
HNC cell lines and primary human IMR90 fibroblasts. β-Lap-50% lethal dose (LD50,
µM), and LD50 for co-treatment with β-lap plus DIC, β-lap plus BAPTA, and β-lap plus
Catalase for HNC cancer cell lines and IMR90 primary fibroblasts with varying NQO1
expression and activity levels.
Supplemental Table 2. Equitoxic doses comparing single to combined treatment
in HNC cells. Equitoxic doses comparing single to combined treatment in HNC cells are
listed in values of equivalent doses (ED) calculated using parameters obtained from
fitting the data from three independent experiments with Carter’s model.
Supplemental Table 3. Individual patient gender, age, subsite, stage, histology
and IHC scoring for NQO1 and Catalase.
Reference:
1. Goswami CP, Nakshatri H: PROGgene: gene expression based survival analysis
web application for multiple cancers. Journal of clinical bioinformatics 2013,
3(1):22.
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