DCLAK11 treatment induces apoptosis in non‐small cell lung cancer cells by inhibiting the mitochondrial apoptosis pathway

DCLAK11 is a novel small molecule tyrosine kinase inhibitor with remarkable effects on cell proliferation and apoptosis in non‐small cell lung cancer cells. Therefore, it is necessary to determine the molecular mechanisms of action of this compound. In this study, we investigated the antitumor effect that DCLAK11 exerts through inhibition of the mitochondrial apoptosis pathway.


INTRODUCTION
Lung cancer is the most prevalent malignancy worldwide and is the leading cause of cancer-related death. 1,2 Non-small cell lung cancer (NSCLC) is the main type of lung cancer, 3 and chemotherapy is an important therapeutic strategy for patients at an advanced stage of this disease. 4 However, the relatively serious side-effects of cytotoxic chemotherapy limit its benefits, and the overall survival of patients with NSCLC undergoing targeted therapies is far from satisfactory. 5 Consequently, more effective treatments are urgently required for patients with NSCLC. DCLAK11 is a novel small molecule tyrosine kinase inhibitor that shows remarkable antitumor activity in NSCLC. 6 In the present study, we aimed to investigate the effects and mechanisms of action of DCLAK11 in inducing apoptosis in the NSCLC cell lines, HCC827 and HCC4006.

Annexin V-PI assay
The

JC-1 assay
The HCC827 and HCC4006 cells were dissociated in pancreatin without EDTA after being incubated with various concentrations of
DCLAK11 treatment markedly inhibited the proliferation of HCC827 and HCC4006 cells in a dose-dependent manner, with an IC 50 value of 10 and 20 nmol/L, respectively (Fig. 1).

DCLAK11 treatment induces apoptosis in NSCLC cells
HCC827 cells were treated with DCLAK11 at various concentrations We also detected the pro-apoptotic effect of DCLAK11 in the

DCLAK11 treatment decreases mitochondrial transmembrane potential in NSCLC cells
As shown in Figure 3, after HCC827 and HCC4006 cells were treated with DCLAK11 for 24 h, damage to the mitochondrial membrane was observed. In HCC827 cells, the proportion of depolarized cells significantly increased from 4.30% to 27.34% with increasing doses of DCLAK11 when compared with that in the control (F = 63.910, P < 0.001). In HCC4006 cells, the proportion of depolarized cells significantly increased from 3.43% to 19.62% in a dose-dependent manner when compared with that in the control (F = 11.831, P = 0.001).

DCLAK11 treatment alters expression of apoptotic proteins
As shown in Figure 4,

DISCUSSION
NSCLC is a malignant tumor that threatens human life, as it has a 5% 5-year survival rate and a median survival of <12 months when diagnosed at the extensive stage. 5 Thus, a compelling need exists for the improved treatment of NSCLC to prolong the survival of patients with NSCLC.
Cell apoptosis plays an important role in balancing cell death and survival, and apoptotic dysfunction might induce carcinogenesis. 7 The mitochondrial pathway, death receptor pathway, and endoplasm reticulum stress pathway are three major apoptotic pathways, among which the first is the most crucial. 8 Apoptosis is reported to be regulated by the Bcl-2 family of proteins, which function upstream of irreversible cellular damage, 9 and regulate the mitochondrial pathway by mediating mitochondrial outer membrane permeabilization. 10 The Bcl-2 family proteins can be divided into two classes: antiapoptotic, which includes Bcl-2, Bcl-xL, Mcl-1, Bcl-w, and Bcl-B; and pro-apoptotic, which includes Bax, Bak, Bok, and the pro-apoptotic BH3-only proteins Bid, Bim, Bad, Bmf, Puma, Noxa, and Bik. 11 In the presence of apoptotic stimuli, such as growth factor deprivation, DNA damage, oxidant stress, or nitric oxide (NO), the expression of Bax and/or the BH3-only proteins Bad, Bid, and Bim increases, and they bind to Bcl-2/Bcl-xL to release Bax/Bak from inhibition. 12 Free Bax and Bak form homo-oligomers, and insert stably into the outer mitochondrial membrane, resulting in increased mitochondrial outer membrane permeabilization, decreased ΔΨm, and release of cytochrome c into the cytoplasm. The released cytochrome c binds with the WD domain of apoptotic protease activating factor-1 (Apaf-1), leading to conformational changes in Apaf-1, followed by deoxyadenosine triphosphate (dATP) binding, and another conformational change in Apaf1 involving exposure and oligomerization of its CARD domain. This step is required for procaspase-9 binding to the exposed CARD domain and efficient assembly of the apoptosome, including cytochrome c, Apaf-1, and caspase-9. The apoptosome assembly results in cleavage of caspase-9, leading to production of the p37 and p19 subunits. Cleaved caspase-9 further processes caspase-3 and caspase-7, leading to apoptosis. [13][14][15] Apoptotic dysfunction is closely related to tumor occurrence and development; 16  is irreversible. 18 The present results showed that DCLAK11 induces mitochondrial membrane depolarization in HCC827 and HCC4006 cells in a dose-dependent manner.
In summary, the findings in the present study show that DCLAK11 induces apoptosis in HCC827 and HCC4006 cells through the mitochondrial pathway by inhibiting the expression of the anti-apoptotic Bcl-2 proteins, enhancing the expression of the proapoptotic Bcl-2 proteins, decreasing the proportion of Bcl-2/Bax, activating caspase-9, and inducing ΔΨm damage. Taken together, these data warrant further evaluation of the potential of DCLAK11 in the treatment of NSCLC.