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. 2024 Aug 9;10(16):e36032.
doi: 10.1016/j.heliyon.2024.e36032. eCollection 2024 Aug 30.

Belamcanda chinensis extract inhibits non-small cell lung cancer proliferation and induces apoptosis via inhibiting the MAPK (Ras/Raf) and Akt pathways

Chong Ma  1   2 , Jingyi Yin  1   2 , Xiao Feng  1   2 , Xin Wang  1   2 , Xiaodie Cao  1   2 , Chen Zhang  1   2 , Rongjie Cui  1   2 , Jingru Wei  1   2 , Xu He  2   3 , Yan Li  2   3 , Li Chen  1   2
Affiliations

Belamcanda chinensis extract inhibits non-small cell lung cancer proliferation and induces apoptosis via inhibiting the MAPK (Ras/Raf) and Akt pathways

Chong Ma et al. Heliyon. .

Abstract

Non-small cell lung cancer (NSCLC) is associated with high mortality and morbidity rates. Despite major progress of treatment of NSCLC over the past few decades, the prognosis of advanced NSCLC is poor, with 5-year survival rates ranging from 2 % to 13 %. Belamcanda chinensis is a traditional Chinese medicine used to promote blood circulation, reduce swelling, heal ulcers, disperse lumps and tumors, and resolve blood stasis. In the present study, the anti-proliferative and pro-apoptotic effects and potential mechanisms of action of Belamcanda chinensis extract (BCE) in SPC-A1 and NCI-H460 NSCLC cells were investigated using MTS, flow cytometry, and western blotting. Also, xenograft model in vivo was established to investigate the anti-NSCLC effects of BCE. The compounds in BCE were quantified using gas chromatography-mass spectrometry (GC-MS). Twenty compounds were found in BCE, and BCE induced cell cycle arrest significantly inhibited the proliferation of NSCLC. Furthermore, BCE was found to induce Cyto C release and the activation of Caspase-3, -8, -9, PARP, ultimately inducing apoptosis in NSCLC cells through both exogenous and endogenous apoptotic pathways (the mitochondrial pathway). BCE also blocked the MAPK (Ras/Raf) and Akt signaling pathways, significantly downregulating the expression of Ras, Raf, Erk1/2, p-Erk1/2, Akt, and p-Akt proteins. Furthermore, BCE significantly inhibited the growth of NSCLC cells SPC-A1 in nude mice and downregulated Ras, Raf, Akt, and p-Akt expression in vivo. The antitumor effects of BCE suggest its potential clinical application in patients with NSCLC, especially in those bearing Ras or Raf mutations.

Keywords: Akt; Apoptosis; Belamcanda chinensis extract; MAPK; Proliferation; Raf; Ras; The non-small cell lung cancer.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
GC-MS chromatogram of the BCE.
Fig. 2
Fig. 2
The effect of BCE on the viability and cell cycle distribution of NSCLC cells. (A) HEK-293T, SPC-A1, and NCI–H460 cells were seeded in 96-well plates at a density of 5 × 103/well and treated with various concentrations (control (0), 25, 50, 100, 200, 400 μg/mL) of BCE for 72 h, and cell viability was examined by MTS assay. (B) BCE triggered cell cycle arrest in NSCLC cells. SPC-A1 and NCI–H460 cells were exposed to different concentrations of BCE (control (0), 40, 80, 160 μg/mL) for 24 h. Then, the cells were stained with PI and the cell cycle distribution was analyzed by flow cytometry.
Fig. 3
Fig. 3
BCE activated apoptosis pathways in NSCLC cells. (A) After treating NSCLC cells with various concentrations of BCE (control (0), 40, 80, 160 μg/mL) for 24 h, apoptotic cells were detected by flow cytometry after double staining with AnnexinV-FITC and PI. (B) Statistical analysis of apoptosis rates through three independent experiments. **, P < 0.01; ***, P < 0.001; one-way ANOVA, post hoc comparisons, Tukey's test. Columns, mean; error bars, SE. (C) BCE led to specific cleavage of PARP and Caspases. NSCLC cells SPC-A1 and NCI–H460 were exposed to escalating concentrations (control (0), 40, 80, 160 μg/mL) of BCE for 24 h or to 160 μg/mL of BCE for various times (control (0), 6, 12, 24 h). Thereafter, PARP and Caspases were examined by Western blot. (D) BCE induced Cyto C release. NSCLC cells were treated with different concentrations of BCE for 24 h, and Cyto C in the cytosolic fraction was measured by Western blot. Fig. 3, Fig. 4A were the same experiment setting, and the Actin bands in Fig. 3, Fig. 4A were the same.
Fig. 4
Fig. 4
Effect of BCE on MAPK (Ras/Raf) and Akt signaling pathways in SPC-A1 and NCI–H460 cells. (A) SPC-A1 and NCI–H460 cells were exposed to various concentrations of BCE (control (0), 40, 80, 160 μg/mL) for 24 h, or to 160 μg/mL of BCE at different times (control (0), 6, 12, 24 h). MAPK (Ras/Raf) and Akt signaling pathway proteins were analyzed by Western blot. (B) NSCLC cells were treated with different concentrations of BCE (control (0), 40, 80, 160 μg/mL) for 24 h. Raf and Ras gene transcription levels were evaluated by RT-qPCR. Fig. 3, Fig. 4A were the same experiment setting, and the Actin bands in Fig. 3, Fig. 4A were the same.
Fig. 5
Fig. 5
Effect of BCE on Ras/Raf mediated by fluorescence bimolecular complementary. (A) After transfection of HEK-293T cells with bimolecular fluorescence complementary vectors Ras-Vc155 and Raf-Vn155 and treatment with gradually increasing concentration of BCE (control (0), 50, 100, 200, 400 μg/mL) for 48 h, fluorescence microscopy was used to observe the bimolecular fluorescence complementary signal. (B) Fluorescence signal reader was used to detect the fluorescence signal in (A). Statistical analysis of fluorescence quantification through three independent experiments. ***, P < 0.001, one-way ANOVA, post hoc comparisons, Tukey's test. Columns, mean; error bars, SE. (C) HEK-293T cells were transfected with Ras-Vc155 and Raf-Vn155 vectors and treated with gradually increasing concentration of BCE (control (0), 40, 80, 160 μg/mL) for 48 h. Thereafter, Western blot was used to detect the expression levels of Ras-Vc155 and Raf-Vn155 proteins.
Fig. 6
Fig. 6
Effect of BCE on the stability of Ras and Raf in proteasome pathway. (A) Western blot was used to detect the combining effects of MG132 (10 μM) and BCE (80 μg/mL) on Ras and Raf proteins in NSCLC cells SPC-A1 and NCI–H460 for 24 h. (B) Analysis of the relative density of Ras and Raf proteins in (A). Statistical analysis of band signal through three independent experiments. *, P < 0.05; ***, P < 0.001, one-way ANOVA, post hoc comparisons, Tukey's test. Columns, mean; error bars, SE.
Fig. 7
Fig. 7
Effect of BCE on the expression of Ras and Raf through apoptosis pathway. (A) After treatment with Z-VAD-FMK (40 μM) or BCE (80 μg/mL) for 24 h, or both, the expression of Ras and Raf proteins were detected by Western blot in NSCLC cells SPC-A1 and NCI–H460. (B) Analysis of the relative density of Caspase-3, Ras and Raf proteins in (A). Statistical analysis of band signal through three independent experiments. ns, no significance; **, P < 0.01; ***, P < 0.001, one-way ANOVA, post hoc comparisons, Tukey's test. Columns, mean; error bars, SE.
Fig. 8
Fig. 8
BCE inhibited the growth of subcutaneous xenografts of SPC-A1 cells in nude mice. (A) The growth curves of subcutaneous xenografts of SPC-A1 cells are shown. The measured tumor size is plotted against the number of days after initiation of BCE treatment. ***, P < 0.001; Comparisons between the two groups were made using the student's t-test. (B) Representative photographs and average weight of tumors isolated after sacrifice of mice. ***, P < 0.001; the student's t-test. (C) The expression of Ras, Raf, Akt and p-Akt proteins in xenograft tumors after BCE treatment was detected by western blotting.
Fig. 9
Fig. 9
Molecular mechanism of BCE inhibiting proliferation and inducing apoptosis of NSCLC cells.
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