Retracted Article: Structural characterization of centipede oligopeptides and capability detection in human small cell lung carcinoma: inducing apoptosis

Abstract

Lung cancer is the most frequent cause of cancer deaths in the world, and smoking is considered as one of the major causes. Small cell lung carcinoma (SCLC) represents a highly malignant and particularly aggressive form, with properties of widespread metastases and poor prognosis. Herein, twenty-five Scolopendra subspinipes mutilans L. Koch Oligopeptides (SSMOs) were isolated and their structures were identified, and the anti-proliferative activity against lung cancer cell lines was evaluated. Results showed that SSMO-5 induced the production of reactive oxygen species (ROS) markedly in NCI-H446 cells. Furthermore, SSMO-5 decreased the mitochondrial membrane potential (MMP) and enhanced the mitochondria-related apoptosis. These results demonstrate that in NCI-H446 cells, the apoptotic and cytotoxic effects of SSMO-5 are mediated by the intrinsic mitochondria-mediated apoptotic pathway, which in turn causes the activation of caspases and increases Bax expression, while decreases Bcl-2 and Bcl-xL expressions and regulates the interaction of p53/MDM2. In conclusion, a ROS-mediated mitochondrial pathway plays an important role in the process of SSMO-5-induced apoptosis against SCLC.

Fig. 1. Structure and MS/MS spectra of SSMO-5 (m/z = 617.3226).

Fig. 2. SSMO-5-induced apoptosis in NCI-H446 cells. Representative scatter diagrams. NCI-H446 cells were pre-treated without the addition of any samples, the control (C) (I), with a dose of 3.84 μM SSMO-5 (II), with a dose of 19.2 μM SSMO-5 (III), and with a dose of 96.0 μM SSMO-5 (IV), respectively, for 48 h. Cells were stained with Annexin-V and PI. The apoptosis of NCI-H446 cells was detected by flow cytometry. The evaluation of apoptosis was via Annexin V: FITC Apoptosis Detection Kit per manufacture's protocol. The quantitative results are also shown. In each scatter diagram, the abscissa represents the fluorescence intensity of the cells dyed by Annexin V, and the ordinate represents the fluorescence intensity of the cells dyed by PI. The lower left quadrant shows the viable cells, the upper left shows the necrotic cells, the lower right shows the early apoptotic cells, and the upper right shows the late apoptotic cells.

Fig. 3. Cell cycle analysis of NCI-H446 cells exposed to SSMO-5. NCI-H446 cells were pre-treated without the addition of any samples, the control (C) (I), with a dose of 3.84 μM SSMO-5 (II), with a dose of 19.2 μM SSMO-5 (III), and with a dose of 96.0 μM SSMO-5 (IV), respectively, for 48 h. C was the control group, without the addition of any tested compounds. Cells were collected, fixed in 70% ethanol, and stained with propidium iodide solution. G0/G1: quiescent state/growth phase; S: initiation of DNA replication; G2/M: biosynthesis/mitosis phases.

Fig. 4. ROS generation and MMP decline of NCI-H446 cells exposed to SSMO-5. (A and B) NCI-H446 cells were pre-treated without the addition of any samples, the control (C) (I), with a dose of 3.84 μM SSMO-5 (II), with a dose of 19.2 μM SSMO-5 (III), and with a dose of 96.0 μM SSMO-5 (IV), respectively, for 48 h. C was the control group, without the addition of any tested compounds. Cells were treated with 19.2 μM SSMO-5 for 0, 2, 4, 8, 12, 24 and 48 h at 37 °C, stained with DCFH-DA for 20 min, and analyzed for fluorescence by flow cytometry. All data are presented as the mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01. (C and D) Flow cytometry analysis of MMP based on JC-1 staining. NCI-H446 cells were pre-treated without the addition of any samples, the control (C) (I), with a dose of 3.84 μM SSMO-5 (II), with a dose of 19.2 μM SSMO-5 (III), and with a dose of 96.0 μM SSMO-5 (IV), respectively, for 48 h. C was the control group, without the addition of any tested compounds. Cells were treated with 19.2 μM SSMO-5 for 0, 2, 4, 8, 12, 24 and 48 h at 37 °C and stained with JC-1. The cells showing a loss of MMP were gated. All data are presented as the mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01. The data shown are representative of three independent experiments.

Fig. 5. Effects of SSMO-5 on the expressions of Bcl-2 family, caspases and p53/MDM2. NCI-H446 cells were pre-treated without the addition of any samples, the control (C) (I), with a dose of 3.84 μM SSMO-5 (II), with a dose of 19.2 μM SSMO-5 (III), and with a dose of 96.0 μM SSMO-5 (IV), respectively, for 48 h. C was the control group, without the addition of any tested compounds. The protein expression levels were measured using western blot. The density of each lane was presented as mean ± standard deviation (SD) for at least three individual experiments. Blots were quantified using Image J software. β-Actin was used as the internal control.