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Merhi et al. J Cancer Metastasis Treat 2021;7:42 https://dx.doi.org/10.20517/2394-4722.2021.80 Page 9 of 16

Figure 4. HF inhibits the production of proMMP-2/9 and VEGF-A by AML cells. (A) Total MMP-2 (n = 11), total MMP-9 (n = 8), and
VEGF-A (n = 13) productions in the 72-h culture supernatants from AML samples untreated or treated with HF (2 μg/mL) were
determined by ELISA. P value was calculated using a Mann-Whitney U-test; *P < 0.05; **P < 0.01. (B) The gelatinolytic activities of
MMP-2/9 were analyzed using zymography (see Figure 2) in the supernatants from four AML cell samples treated or not with HF
(2 μg/mL, 72 h). (C) PCR analyses of MMP-2, MMP-9, VEGF-A, and β2-microglobulin transcripts from 5 AML samples treated or not
with HF (2 μg/mL, 24 h). (D) Correlations between the levels of HF-mediated release of MMP-2/9 or VEGF-A and HF-mediated death
in AML cells (n = 11 for MMP-2; n = 8 for MMP-9; and n = 13 for VEGF-A). The percentage of HF-mediated AML cell death was
determined as described in Figure 3. The percentage of HF-mediated release was determined by subtracting the percentage of
MMP/VEGF-A release in the absence of HF from the percentage of MMP/VEGF-A release in the presence of HF, and then dividing by
the percentage of MMP/VEGF-A release in the absence of HF × 100. Spearman’s correlation coefficient (r) and the P-value are shown.

the untreated AML group and the HF-treated group. The levels of released MMP-2/9 and VEGF-A were
not correlated with basal apoptosis in the group of unstimulated AML cells (P > 0.05). In contrast, we
identified in the group of HF-treated AML cells a negative correlation between HF-mediated proteins’
release and HF-mediated apoptosis [Figure 4D]. HF-mediated AML cell death was significantly (negatively)
correlated with the release of MMP-2 (r = -0.856, P = 0.0016) and MMP-9 (r = -0.881, P = 0.0072) but not
with that of VEGF-A (r = -0.542, P = 0.0588) [Figure 4D]. These results indicate that HF induces the release
of MMP-2/9 and VEGF-A in AML cells.