4. Discussion
Currently, anti-neoplastic drugs are more or less deficient in terms of
weak targeting, toxic side effects, and drug resistance [39]. The
search for novel antitumour drugs with low side effects and high
targeting is one of the important topics of research in the field of
medicinal chemistry. Plant endophytic fungi are an important source of
novel antitumour compounds due to their unique ecological niche, which
harbours unique and novel compound structures. A strain ofAlternaria alstroemeria was discovered and isolated by our group,
and its crude extract showed a strong inhibitory effect on A549
[24]. To investigate the molecular basis of the antitumour activity
of this strain, the global regulator of secondary metabolism viz AaLaeA
was overexpressed. It was found that AaLaeA OE29crude extract inhibited A549 significantly more than WT and increased
the total apoptosis rate by 1.56-fold and AaLaeA mediated the antitumour
activity of the crude extract [24]. The function of LaeA has been
reported to be conserved in previous studies and is involved in the
biosynthesis of a variety of fungal secondary metabolites [13].
Overexpression of LaeA or its homologs in Penicillium chrysogenum[15], Chaetomium globosum [17], and Aspergillus
nidulans [40] all resulted in a large increment in fungal secondary
metabolites; and these reports are consistent with the results of our
group’s previous studies. Interestingly, when we screened for AaLaeA
overexpression strains, we found a transformant numbered 26 with 10-fold
higher expression of AaLaeA than WT, but with a significant loss of
antitumour activity. To clarify whether the insertion was caused by a
disruption of the relevant gene at the insertion site, the transformant
was subjected to genome re-sequencing, which revealed that no coding
gene was present upstream or downstream of the insertion site and that
the insertion was a nonsense insertion. It is hypothesized that the
AaLaeA-mediated loss of antitumour activity is not directly correlated
with the insertion site, but may be quantitatively related to the
expression of AaLaeA itself. Metabolomic analysis revealed thatAaLaeA OE26 contained significantly lower levels
of secondary metabolites of antitumour activity compared to A.
alstroemeria wild type. These differential metabolites were carboxylic
acids and their derivatives, fatty acids, phospholipid glycerols,
benzene and its substituents, flavonoids, phenols, indole derivatives,
and other intermediate derivatives which were reported to have the
potential for antitumour activity. In particular, the flavonoids were
more significantly down-regulated, with the most significant
down-regulation and the largest fold difference being in the flavonoid
epicatechin gallate. In combination with comparative transcriptome
analyses, a significant down-regulation of the gene encoding the
FAD-binding domain-containing protein (Fla1) was found; Fla1 belongs to
the FAD-binding domain superfamily and is a
flavoenzyme that is widely found in
nature [18]; It is capable to catalyze a variety of biochemical
reactions and has been widely used as a biocatalyst for a variety of
reactions, and many biochemical processes take advantage of the
versatility of flavoenzymes and extremely such as flavin cofactors
[19, 20]. It is hypothesized that AaFla1 may be negatively regulated
by AaLaeA, thereby affecting the synthesis of the antitumour compounds
in A. alstroermeria . To further verify the relationship between
AaLaeA and AaFla1 genes, the transcript levels of Fla1 in wild-type andAaLaeA OE26 strains were analyzed by qRT-PCR.
The results showed that overexpression of AaLaeA resulted in a
significant down-regulation of Fla1 transcription. In contrast,
overexpression of Fla1 caused a significant increment in the antitumour
activity of the strain, with a reduction in IC50 from
445 μg/mL to 368 μg/mL and a significant 7-fold increment in apoptosis.
Antitumor-related compounds have been reported to accumulate in Fla1
overexpressing transformants, for example, Quercetin [26],
Strychnopentamine [27], Gitogenin [28], Rhodioloside [29],
Liensinine [30], L-Selenomethionine [31], Compactin [32],
Tonantzitlolone B [33], Ginsenoside Rg2 [34], Campesterol
[35], Pristimerin Derivative [36], Cinobufagin [37] and
Anisomycin [38], with the flavonoid Quercetin being the most
significantly up-regulated. This result is in contrast to the
significant reduction in flavonoid content ofAaLaeA OE26, which also confirms in terms of
compound accumulation that AaFla1 is negatively regulated by AaLaeA, and
therefore affecting the antitumour activity of the strain.