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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: Therapeutic evaluation of palbociclib and its compatibility with other chemotherapies for primary and recurrent nasopharyngeal carcinoma

Fig. 5

Involvement of autophagy-related pathways in the enhanced cell death of NPC cells under combined treatment with palbociclib and SAHA. a. Venn diagram of the numbers of upregulated DEGs in the three NPC cell lines after combination treatment with palbociclib + SAHA. The number in the central region denotes the number of overlapping genes in all three cell lines. A total of 914 genes were commonly upregulated in all three cell lines treated with palbociclib + SAHA relative to their vehicle-treated counterparts. b. The top 20 enriched pathways among the 914 DEGs were identified from the CPBD database and are listed in the order of p-value, which depended on the numbers and fold changes of the genes. Upregulated genes were enriched in autophagy or its related cellular functions (highlighted in red rectangles). c. Detection of the autophagy marker, LC-3II, in the C666–1, C17, and NPC43 cell lines treated with the vehicle, palbociclib (15 μM), SAHA (5 μM), or palbociclib + SAHA. The numbers above the blots indicate the normalized grayscale values of LC3-II. d. Autophagic flux analysis of C666–1 cells treated with palbociclib, SAHA, or palbociclib + SAHA and chloroquine (CQ), an inhibitor of autophagic flux (25 μM), for 24 h. Lower panel: Bar chart demonstrating the fold changes in LC3-II relative to the vehicle control. e. Viability of C666–1 cells subjected to the same treatment as in Fig. 5d for 24, 48, and 72 h. f. Knockdown of the autophagy-related proteins BECN1 and ATG5 via siRNAs partially reversed the cytotoxic effects in C666–1 cells treated with palbociclib and SAHA for 24 h. Cell viability was tested using the resazurin assay. Corresponding verification of the siRNA-mediated knockdown efficiency is presented in Supplementary Figure S14. g. Detection of the autophagy level in NPC43 cells using the Autophagy Tandem Sensor GFP-LC3B kit. The cells were treated with palbociclib and SAHA for 24 h as described in Fig. 5c. The presence of GFP-LC3-II–positive autophagic vacuoles in the NPC cells was visualized using a Carl Zeiss LSM 880 confocal microscope. h. Combined treatment upregulated the turnover of LC-3II in C666–1 the tumors in vivo relative to the other treatment groups. Left panel: Western blot analysis of LC-3I and LC-3II in tumors excised from mice in different treatment groups. Middle panel: Bar chart of the LC3-I and LC3-II levels relative to the corresponding GAPDH level in each treatment group, as detected in the left panel. Right panel: Bar chart of the LC3-II/LC3-I ratio in each treatment group as detected in the left panel. P < 0.0001 ****, p < 0.001***, p < 0.005**, p < 0.01*

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