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