Vanilla planifolia stems extracted by ethanol reduces viability and colony formation and induces autophagy in GBM cells.
In this study, three GBM cell lines were used to evaluate the antitumor activities of ethanol extract and water extract from vanilla planifolia, including patient-derived temozolomide (TMZ)-resistant GBM P#5 TMZ-R cells, T98G cells, and U-87 MG cells. First, P#5 TMZ-R cells were treated with the pods, leaves, and stems of vanilla planifolia crude extracts of ethanol and water at 50 and 100 ng/µl. Figure 1A shows that the ethanol extract and water extract of vanilla planifolia pods and leaves only had slightly inhibitory effects. Interestingly, ethanol extract of vanilla planifolia stem (VAS) significantly reduced the cell viability of P#5 TMZ-R at 50 and 100 ng/µl, but the water extract of vanilla planifolia stems did not inhibit the cell viability. Then, to explore the IC50, the cells were further treated with VAS at 50, 100, 150, and 200 ng/µl. Figure 1B shows that VAS effectively inhibited the viability of P#5 TMZ-R cells (IC50: 137 ng/µl), T98G cells (IC50: 150 ng/µl), and U-87 MG cells (IC50: 168 ng/µl). Furthermore, 200 ng/µl VAS significantly inhibited colony formation in P#5 TMZ-R, T98G, and U-87 MG cells (Fig. 1C and D). To further examine the cell death pathway after 200 ng/µl VAS treatment for 72 h, our data showed that MAP1LC3-II conversion was a more robust readout than the control in P#5 TMZ-R, T98G, and U-87 MG cells (Fig. 2A and B). Taken together, these data indicate that VAS treatment induced autophagy and reduced the viability and colony formation of GBM cells.
VAS led to 4248 differentially expressed genes in GBM cells.
Next, to verify which VAS-mediated genes led to cell death, RNA-seq was used to measure mRNA expression after VAS treatment. Based on our findings, the cells were treated with 200 ng/µl VAS and analyzed by RNA-seq. The transcripts were compared between 200 ng/µl VAS and the vehicle control. The volcano plot and MA plot of DEGs between the two groups are shown in Fig. 3A and B. Compared with the vehicle control, 1972 DEGs were upregulated (p < 0.05, log2-fold change > 1), and 2276 DEGs were downregulated (p < 0.05, log2-fold change < -1). Then, we used KEGG enrichment and GO to determine the functional annotation of these genes to investigate the involvement of expressed DEGs after VAS treatment in biological process (BP), molecular function (MF), and cellular component (CC). KEGG analysis showed that the cell cycle, apoptosis, cellular senescence, autophagy, and lysosome were the top five enriched terms (Fig. 3C). GO analysis showed that in BP enrichment analysis, spindle organization, DNA replication, and macroautophagy were the top three enriched terms (Fig. 3D). In the MF category of GO analysis, small GTPase binding, GTPase binding, ubiquitin-like or ubiquitin protein ligase binding, and cadherin were the top enriched terms (Fig. 3E). The terms associated with CC may be linked with chromosomal region, nuclear envelope, and spindle (Fig. 3F). Taken together, these enrichment analysis results implied that VAS affected critical functions and pathways for survival and proliferation in GBM cells.
To further explore the most important DEG clustering of GBM survival after VAS treatment, we constructed a protein‒protein interaction (PPI) network. Eight-nine downregulated DEGs with log2-fold change < -3 were analyzed by the STRING database, and 16 hub DEGs were correlated, including TNMD, MATN1, RLBP1, IGFBP1, PCP4, DHRS9, GNAT2, C2CD4C, ELFN2, GDA, AQP5, SKAP1, HOPX, PLCB2, FA2H, and RGS8 (Fig. 4A). The fold changes in these hub DEGs determined by RNA-seq are shown in Fig. 4B. Similarly, nine upregulated DEGs in twenty-three DEGs with log2-fold change > 5 showed correlation using STRING analysis, including CXCL10, IL36RN, MX2, EGR1, RSAD2, CCL5, IFI44L, CCL20, and HMOX1 (Fig. 4C). The fold changes in these upregulated DEGs determined by RNA-seq are shown in Fig. 4D.
VAS significantly downregulated 14 DEGs and upregulated 9 DEGs by qPCR in GBM cells.
To further validate the expression of these hub DEGs, a qPCR experiment was performed in P#5 TMZ-R cells and U-87 MG cells. Figure 5A shows that the mRNA expression levels of TNMD, MATN1, RLBP1, IGFBP1, PCP4, DHRS9, GNAT2, C2CD4C, ELFN2, AQP5, SKAP1, HOPX, FA2H, and RGS8 were significantly reduced after VAS treatment in both P#5 TMZ-R cells and U-87 MG cells. Moreover, VAS also significantly induced the mRNA expression of CXCL10, IL36RN, MX2, EGR1, RSAD2, CCL5, IFI44L, CCL20, and HMOX1 in both P#5 TMZ-R cells and U-87 MG cells. (Fig. 5B). Taken together, these findings implied that VAS could influence the expression of numerous genes and mediate cell death in GBM cells.