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Page 10 of 16 Merhi et al. J Cancer Metastasis Treat 2021;7:42 https://dx.doi.org/10.20517/2394-4722.2021.80

DISCUSSION
Enhanced angiogenesis participates in the progression of AML, and pharmacological targeting of pro-
angiogenic proteins (including MMPs and VEGF-A) might be a possible antileukemic strategy. HF exhibits
antiangiogenic and proapoptotic activities towards various cancer cells [15,19,29,30] . AML cells abnormally
proliferate and escape apoptosis. Our present study provides evidence that primary blood AML cells co-
express proMMP-2, proMMP-9, and VEGF-A proteins. The major finding of our study indicates that HF is
able to inhibit two biological events related to AML progression and angiogenesis, i.e., survival and
secretion of MMP-2/9 and VEGF-A in AML patients’ cells.

Peripheral blood blasts from AML patients express detectable levels (transcript and secreted protein) of
[45]
MMP-2, MMP-9, and VEGF-A. As previously reported for BM AML blasts , circulating AML cells release
the proforms of MMP-2 (72 kDa) and MMP-9 (92 kDa) proteins. An additional 85 kDa form of MMP-9 is
observed in some AML samples (independently of the FAB subtype). A previous study reported that BM
AML blasts with the subtypes M4/M5 express an ≃ 85 kDa MMP-9 at their cell surface . Breast cancer cells
[58]
[59]
express an 85-kDa MMP-9 devoid of complex carbohydrates, which might result from deglycosylation .
Interestingly, active MMP-3 and active MMP-13 can cleave proMMP-9 to generate an 86-kDa
intermediate [60,61] . The origin of this MMP-9 variant and its role in AML remain to be defined.

We observed no intercorrelations among the levels of MMP-2, MMP-9, and VEGF-A secreted by AML
cells, likely reflecting their variable expression both among and within individual AML cells. MMP-2
transcription is mainly regulated by signal transducer and activator of transcription 3 (STAT3) [62,63] , whereas
MMP-9 transcription is regulated via NF-κB, AP-1, and SP-1 . VEGF transcription can be regulated by
[64]
STAT3 and HIF [62,63,65] . Our data strongly suggest that the heterogeneity found in the release profiles of
MMP-2/9 and VEGF-A proteins may be related to the distinct gene regulations of MMP-2/9 and VEGF in
AML cells. Moreover, the released levels of MMP-2/9 and VEGF-A do not appear to depend on the
differentiation stage of AML cells. Our data agree with studies in which the levels of MMP-2 (plasma),
MMP-9 (BM), or VEGF-A (BM) and FAB subtypes are not associated [39,40,45,46,66] . In contrast, other studies
have shown an association between MMP-9 or VEGF-A (BM) and AML-M4/M5 [43,46] , as well as between
VEGF-A (BM) and AML-M0 . Larger studies are mandatory to fully address this question.
[42]
We previously showed that HF exhibits proapoptotic activities in AML cell lines of distinct FAB
phenotype . In particular, HF did not activate the enzymatic activities of caspase-3/8/9 directly (B. Bauvois,
[26]
unpublished results) but promoted caspase-mediated apoptosis involving BAD and Noxa activation in AML
[26]
U937 (M5) cells . BAD is a direct downstream target of Akt1 . We previously presented the first evidence
[67]
that HF, by directly inhibiting Akt-1 kinase activity, prevents BAD phosphorylation, which in turn activates
its proapoptotic role in U937 cells . Primary AML cells overexpress Akt1 . Activated Akt signaling
[26]
[67]
protects AML cells from apoptosis . In this study, the proapoptotic effect of HF in AML cell lines is
[67]
confirmed in primary AML cells, independently of the latter’s FAB subtype; HF-mediated AML cell death
occurred in 33 of the 35 AML samples tested, accompanied by caspase-3 activation. Whether HF activates
the proapoptotic function of BAD through Akt1 inhibition in primary cells remains to be confirmed. HF
exerts selective cytotoxicity on AML cells but not on normal PBMCs and monocytes. Importantly, BM
AML cells appear as sensitive as circulating AML cells toward the apoptotic effect of HF, suggesting that HF
might be of interest for the therapy of AML through its potential capacity to eradicate BM leukemic stem
cells. Moreover, the induction of apoptosis by 2 μg/mL of HF parallels the inhibition of secretion of MMP-
2/9 and VEGF-A by HF at the same concentration. Suppression of released MMP-2/9 and VEGF-A proteins
by HF does not appear correlated with their transcript levels, suggesting a post-transcriptional inhibition.
Our observations strongly suggest that HF induces simultaneously: (1) suppression of MMP-2/9 and VEGF-