Discussion
More
and more studies have shown that NCTD can induce apoptosis in a variety
of tumor cells. However, so far, whether NCTD has an effect on non-small
cell lung cancer (NSCLC) cells is unknown. In this study, we used
different concentrations of NCTD to observe the proliferation of A549
cells and explore its molecular mechanism. MTT analysis showed that the
proliferation ability of A549 cells decreased with the increase of NCTD
concentration. The colony forming ability experiments showed that the
increase in NCTD concentration was inversely related to the number of
cell clones. These data indicate that NCTD has an inhibitory effect on
cell proliferation and activity (Figure 1B, 1C). The cell cycle plays an
important role in the development of malignant tumors, and cycle changes
are a major feature of malignant tumors [10]. Previous studies have
shown that tumor cell development may be related to changes in the
expression of cell cycle-related regulatory genes (including
cyclin-dependent cyclin-related genes), thereby blocking cells in the G2
/ M phase (CDKIs; such as p21) [11,12]. Then we used flow cytometry
to find that as the drug concentration increased, the proportion of
cells in the G2 phase of the tumor cells also increased. Figure 2A,
which shows that NCTD downregulates cyclin D3 and cyclin E2 and
upregulates cyclin A and cyclin B (Figure 2C ), the results are
consistent. These data indicate that NCTD can inhibit the proliferation
of A549 cells by blocking the G2 phase. p21 is a member of the Cip / Kip
family and can regulate CDK and cyclin, thereby affecting the transition
from G2 to M phase. Our findings (Figure 2B, C), even though cyclin A
and cyclin B (G2 phase-related proteins) increase, p21 still inhibits
the activity of the cdc2 / cyclin B complex, so we conclude that NCTD
increases the expression of p21 Inducing G2 cell cycle arrest, Akt may
also inhibit p21 expression through its phosphorylation and MDM2
activation and subsequent p53-mediated down-regulation of p21
transcription. Our results indicate that the expression of p-AMPK is
increased and the expression of p-Akt is decreased (Figure 6A),
indicating that NCTD activates AMPK, inhibits Akt, up-regulates p21
expression and inhibits the active B complex of cdc2 / cyclin, thereby
stopping the cell cycle. G2 / M.
Apoptosis is the main form of tumor cell death. In our study, flow
cytometry analysis of NCTD-treated tumor cells was found to have a
positive correlation between the apoptotic rate and NCTD concentration
(Figure 3A, C ). Apoptosis is associated with cytochrome c
release, caspase-3 activation, and PARP cleavage [13]. Enhanced
permeability of the outer mitochondrial membrane, reduced mitochondrial
membrane potential, mitochondria release pro-apoptotic molecules such as
cytochrome C to the cytoplasm, thereby activating caspase-9, causing a
cascade reaction leading to cell death [14,15]. In this study, we
found that NCTD reduced the ΔΨm point. (Figure 3B, D). Bcl-2 and Mcl-1
decreased in the Bcl-2 family, while Bax and Bcl-xL remained unchanged,
resulting in an increase in the ratio of Bax / Bcl-2 and Bax / Mcl-1
(Figure 4A). In the caspase family into caspase-3, the activation of
caspase9 (Figure 4B ) activates caspase-3 to cleave PARP and
cause changes in morphology and biological apoptosis. Because the
caspase-8 protein changes are not obvious, we speculate that
NCTD-induced A549 cell apoptosis does not involve an exogenous apoptotic
pathway (Figure 4B ).
Autophagy mainly protects cells from external stimuli and plays a role
in regulating and controlling the internal environment of the cell
[16], but excessive autophagy consumes components in the cell and
causes cell death[17]. Previous studies have shown broad prospects
for the treatment of malignant tumors through autophagy. In this study,
we demonstrated that NCTD treatment can increase the conversion of LC3-I
to LC3-II, reduce the expression of p62 (Figure 5A), and increase the
spotted LC3 in the A549 cytoplasm (Figure 5C). It is shown that NCTD
induces autophagy in A549 cells.Mitochondria is a mitochondrial recovery
process that mainly promotes cell survival, but may also lead to cell
death under conditions of excessive injury. Mitochondrial depolarization
is an early event in which physiological function is affected in
mitochondria [18]. The function of mitochondrial phagocytosis has
been studied and is thought to be an early manifestation of cellular
autophagy. Mitochondrial autophagy induces mitochondrial phagocytosis
[19]. TOM20 protein is a mitochondrial outer membrane marker
protein, so changes in its expression level can reflect mitochondrial
autophagy status [20]. It was found in the study that NCTD induced a
significant decrease in ΔΨm in A549 cells (Figure 3B, 3D). After NCTD
treatment of cells, the expression of TOM20 protein was reduced (Figure
5B) and characteristic aggregation of mitochondria was triggered.
(Figure 5D). The above data indicates that NCTD induced mitochondrial
phagocytosis in A549 cells.
Previous studies have shown that the Akt / mTOR signaling pathway plays
an important role in cell proliferation, and the activation of this
pathway is related to the occurrence and development of various
malignancies[20]. This study found that the antitumor drug NCTD
induces early autophagy and late apoptosis of tumor cells through the
Akt / mTOR signaling pathway [21]. mTOR is a key factor in autophagy
activation.[22]. The upstream mTOR signaling pathway plays an
important role in cell growth and cell cycle, mainly through the PI3K /
Akt / mTOR pathway and the independent PI3K / Akt pathway. Akt belongs
to the serine / threonine protein kinase subfamily and is a kinase
upstream of AMPK. AKT inhibits AMPK and activates mTOR through the
AMPK-TSC-MTOR pathway [23]. Previous studies have shown that NCTD
can activate AMPK in mammalian cells[24]. AMPK can induce autophagy
directly through the ULK1 protein or indirectly through inhibition of
the mTOR signaling pathway [25]. In this study, NCTD reduced the
expression of p-Akt, increased the expression of p-AMPK and ULK1, and
suppressed the expression of mTOR, suggesting that NCTD promoted
autophagy in A549 cells (Figure 6A). C-jun-N-terminal kinase (JNK) is a
mitogen-activated protein kinase family member that can be activated by
a variety of external factors, such as radiation, bacteria, drugs,
hypoxia, and endoplasmic reticulum stress. JNK signal transduction in
cell proliferation, differentiation, apoptosis, ROS accumulation
[26]. JNK-mediated 14-3-3 protein phosphorylation promotes its
dissociation from Bax and translocation to mitochondria, leading to Cell
death. Phosphorylation of JNK-mediated Ser70 of Bcl-2, Thr47 and Thr115
of Bcl-xL, and Ser121 and Thr163 of Mcl-1 can inactivate these
anti-apoptotic proteins in response to cellular stress. Similarly,
JNK-mediated phosphorylation of Ser128 in Bad, Ser65 in Bim, Thr56 in
BimL, and Thr67 in Bax promote these proapoptot-ic proteins[26]. In
this study, we found that NCTD activates JNK and downstream c-jun
proteins, promoting apoptosis and autophagy (Figure 6B).
As shown in Figure 7, NCTD treatment activated Akt, then up-regulated
p21 and inhibited the activity of cdc2 / cyclin B, and finally induced
cell cycle arrest in the G2 / M phase. Inhibits p-Akt, downregulates
mTOR, up-regulates ULK1, and promotes autophagy. AMPK activation also
inhibits mTOR expression and increases ULK1, thereby promoting cellular
autophagy. On the other hand, inhibition of Akt and activation of JNK
and AMPK together regulate the inhibition or activation of members of
the Bcl-2 family. The negative regulation of Bcl-2 and Mcl-1 and the
reduction of mitochondrial membrane potential induce mitochondrial
autophagy. The above changes cause the activation of the caspase family,
leading to the cleavage of PARP and ultimately the apoptosis of A549
cells. Mitochondrial activation is also involved in autophagy and
apoptosis induced by drug action on A549 cells. In general, NCTD can
induce tumor cell autophagy and apoptosis by regulating the Bcl-2
family, inducing mitochondrial dysfunction, activating the caspase
family, inhibiting the PI3K / Akt / mTOR pathway, and activating the
AMPK / ULK1 and JNK signaling pathways ,NCTD causes cytotoxicity to
trigger A549 cell apoptosis