Page 26 - Read Online

Merhi et al. J Cancer Metastasis Treat 2021;7:42 https://dx.doi.org/10.20517/2394-4722.2021.80 Page 5 of 16

Measurement of MMP-2/9 gelatinolytic activity by zymography
Analysis of MMP-2/9 activities in culture supernatants was carried out in 7.5% (w/v) SDS-polyacrylamide
[52]
gels containing 0.1% gelatin (w/v), as described in . Gelatinolytic activities of MMPs were detected as
transparent bands on the background of Eza-blue stained gelatin. The bands were acquired in a
densitometer (Oncor).

ELISA analysis
Total MMP-2 (pro and active forms), total MMP-9 (pro and active forms), and VEGF-A (containing 125
amino acid residues, VEGF ) contents in culture supernatants were determined using commercial ELISA
125
kits provided by R&D (Abingdon, UK). Controls included culture medium alone incubated under the same
conditions. Detection levels for MMP-2/9 was 1 ng/mL and for VEGF-A 5 pg/mL.

Statistics
Statistical analyses were performed using GraphPad Prism software (version 7.0, GraphPad Software, La
Jolla, CA, USA). Groups were compared using Mann-Whitney tests or unpaired or paired Student’s t-tests.
Correlations between variables were tested by calculating Spearman’s coefficient (r) and the P-value ≤ 0.05
was considered statistically significant. All tests were two-tailed for greater stringency.

RESULTS
Expression of proMMP-2, proMMP-9, and VEGF-A in primary AML cells
We first examined the levels of transcripts of MMP-2/9 and VEGF-A in leukemic blood cells from 15 AML
patients with different FAB subtypes M0, M1, M2, M4, and M5 (FAB M3 is not represented because of its
low frequency in the cohort). Representative examples of PCR products are shown in Figure 2A. The PCR
products for MMP-2/9 and VEGF-A were detected at various degrees in all AML samples tested (Figure 2A
and data not shown). MMP-2 and VEGF-A transcripts were detected in all tested samples, whereas MMP-9
transcript was detected in 12 of the 15 tested samples (Figure 2A and data not shown). The transcripts
patterns showed no associations with the FAB subtype (Figure 2A and data not shown). As exemplified in
Figure 2B, zymography analysis of the supernatants of AML cells (72 h of culture) showed the presence of
proMMP-9 and proMMP-2 proteins at 92 and 72 kDa, respectively [Figure 2B], whereas the active forms of
MMP-9 (at 82 kDa) and MMP-2 (at 65 kDa) were not detected [Figure 2B]. In some cases, an intermediate
≃ 85 kDa MMP-9 form was seen [Figure 2B]. The release of these proteins by primary AML cells in vitro
and under basal conditions was quantified in ELISAs: the mean (range) of total MMP-2, total MMP-9, and
VEGF-A concentrations (after a 72 h of culture) released by AML cells were, respectively, 4.44 ng/mL
6
(1-13 ng/mL) for 10 cells, 16.04 ng/mL (1-51 ng/mL) for 10 cells and 57.71 pg/mL (5-288 pg/mL) for 10
6
6
cells [Figure 2C] and were not correlated with the FAB subtype (P > 0.05). Next, we tested for associations
between the levels of released MMP-2, MMP-9, and VEGF-A. No correlations were found for MMP-2 and
MMP-9 (r = 0.2859, P = 0.1483), MMP-2 and VEGF-A (r = 0.1046, P = 0.6867), or MMP-9 and VEGF-A (r =
0.1817, P = 0.4811) [Figure 2D].

HF induces apoptosis in primary AML cells independently of the FAB subtype
We assessed the effects of HF on the viability of blood PBMCs obtained from 37 AML patients. Cell death
was assessed by determining phosphatidylserine exposure at the cell surface (using annexin-V-FITC
binding) and cell membrane disruption (using propidium iodide labeling). Initial studies showed that HF’s
optimal effects in inducing death of AML cells occurred in a time-dependent manner (24-72 h).
Consequently, the lethal effect of HF at 72 h was investigated in all subsequent experiments. As exemplified
in Figure 3A, the proportion of total annexin-V cells (dead cells) by 72 h enhanced after treatment with
+
increasing doses of HF (1.4, 2, and 3 μg/mL) compared to control (untreated) experiments. Accordingly,
light microscopy observations revealed that AML cell treatment with HF led to a deterioration of the cell