Crebanine Induces Cell Death and Alters the Mitotic Process in Renal Cell Carcinoma In Vitro

Abstract

Advanced renal cell carcinoma (RCC) has a poor prognosis; this drives the exploration of alternative systemic therapies to identify more effective treatment options. Recent research has revealed that crebanine, an alkaloid derivative of the Stephania genus, induces apoptotic effects in various cancers; however, a thorough investigation of the role of crebanine in RCC has not been conducted thus far. For this study, we evaluated tumor cell viability, clonogenicity, cell-cycle distributions, morphological changes, and cell mortality with the aim of exploring the antitumor effects of crebanine in RCC. Furthermore, we compared gene and protein expressions using RNA sequencing analysis and Western blotting. The findings indicated that crebanine significantly inhibited RCC colonies and caused G1-phase cell-cycle arrest with sub-G1-phase accumulation, thus leading to suppressed cell proliferation and cell death. In addition, Hoechst 33342 staining was used to observe apoptotic cells, which revealed chromatin condensation and a reduction in the nuclear volume associated with apoptosis. Further, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that differentially expressed genes are involved in the initiation of DNA replication, centrosome duplication, chromosome congression, and mitotic processes in the cell cycle along with signaling pathways, such as I-kappaB kinase/NF-kappaB signaling, Hippo signaling, and intrinsic apoptotic pathways. Consistent with GO and KEGG analyses, increased levels of cleaved caspase-3, cleaved caspase-7, and cleaved PARP, and decreased levels of cIAP1, BCL2, survivin, and claspin were observed. Finally, the expressions of G1/S phase transition cyclin D1, cyclin E/CDK2, and cyclin A2/CDK2 complexes were downregulated. Overall, these findings supported the potential of crebanine as an adjuvant therapy in RCC.

Keywords: Stephania venosa; aporphine alkaloid; kidney cancer; natural product; systemic treatment.

Figure 1

Crebanine inhibits RCC cell growth in a potent apoptotic manner. (A) Crebanine reduced the cell viability of the 786-0, A498, and Caki-1 cells, as determined by the MTT assay, where 25 µM, 50 µM, 100 µM, and 200 µM of crebanine and DMSO were used in the MTT assay, respectively. (B) While evaluating the growth changes in 786-0, A498, and Caki-1 cells in 50 µM and 200 µM crebanine, the latter was found to inhibit the formers’ colony formations. (C,D) Crebanine increased RCC cells arrested in the sub-G1 phase. Cell-cycle distributions were evaluated by flow cytometry. Cell-cycle distributions were shown for the 786-0, A498, and Caki-1 cells. Data were shown as the mean ± SD (* p < 0.05, ** p < 0.01; *** p < 0.001).

Figure 2

Crebanine promotes apoptosis in RCC cell lines. (A) Annexin V/PI staining was performed with flow cytometry in 786-0, A498, and Caki-1 cells, which were treated with crebanine (0 µM, 50 µM, and 200 µM) for 48 h. (B) The proportions of Annexin^+/− PI^+/− distribution of 786-0, A498, and Caki-1 cells were treated with crebanine (0 µM, 50 µM, 200 µM) for 48 h, which are depicted as bar charts. (C) Apoptotic cells with morphological changes in nuclei increased in response to crebanine treatment. 786-0, A498, and Caki-1 cells were treated with crebanine (0, 50, and 200 µM) for 24 h. (D) The quantity of apoptotic cells in response to treatment is depicted in percentage units. Data are presented as the mean ± SD (*** p < 0.001).

Figure 3

Differential gene expression analysis of renal cell carcinoma. (A,B) Cell-cycle-related and apoptosis-related GO biological process bar chart. The p-value is depicted in color. (C,D) Cell-cycle-related and apoptosis-related GO biological processes are presented by the dot chart. The p-value is depicted in color. The size of the dots reflects the gene ratio. (E,F) Cell-cycle-related and apoptosis-related GO biological processes are depicted by the heat map. Log2-fold change is depicted in color.

Figure 4

Differential gene expression analysis of renal cell carcinoma. (A,B) KEGG analysis of the models. (C) Gene heat map of the model. Downregulated genes are depicted in red. Upregulated genes are depicted in blue.

Figure 5

Validation of apoptosis and cell-cycle-related proteins via Western blotting. The protein expression changed within 24 h. The 50 µM or 200 µM crebanine treatments on RCC cells were measured by Western blotting, including (A) apoptosis-related proteins and (B) cell-cycle-related proteins.