Janta S et al. (2023), Holothurin A Inhibits RUNX1-Enhanced EMT in Met...

ECB-ART-51339

Mar Drugs 2023 Jun 03;216:. doi: 10.3390/md21060345.

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Holothurin A Inhibits RUNX1-Enhanced EMT in Metastasis Prostate Cancer via the Akt/JNK and P38 MAPK Signaling Pathway.

Janta S , Pranweerapaiboon K , Vivithanaporn P , Plubrukarn A , Chairoungdua A , Prasertsuksri P , Apisawetakan S , Chaithirayanon K .

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Due to the challenge of prostate cancer (PCa) management, there has been a surge in efforts to identify more safe and effective compounds that can modulate the epithelial-mesenchymal transition (EMT) for driving metastasis. Holothurin A (HA), a triterpenoid saponin isolated from Holothuria scabra, has now been characterized for its diverse biological activities. However, the mechanisms of HA in EMT-driven metastasis of human PCa cell lines has not yet been investigated. Moreover, runt-related transcription factor 1 (RUNX1) acts as an oncogene in prostate cancer, but little is known about its role in the EMT. Thus, the purpose of this study was to determine how RUNX1 influences EMT-mediated metastasis, as well as the potential effect of HA on EMT-mediated metastasis in endogenous and exogenous RUNX1 expressions of PCa cell lines. The results demonstrated that RUNX1 overexpression could promote the EMT phenotype with increased EMT markers, consequently driving metastatic migration and invasion in PC3 cell line through the activation of Akt/MAPK signaling pathways. Intriguingly, HA treatment could antagonize the EMT program in endogenous and exogenous RUNX1-expressing PCa cell lines. A decreasing metastasis of both HA-treated cell lines was evidenced through a downregulation of MMP2 and MMP9 via the Akt/P38/JNK-MAPK signaling pathway. Overall, our approach first demonstrated that RUNX1 enhanced EMT-driven prostate cancer metastasis and that HA was capable of inhibiting the EMT and metastatic processes and should probably be considered as a candidate for metastasis PCa treatment.

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	Figure 1. Chemical structure of holothurin A.
	Figure 2. The effect of HA on the viability of human PCa cell lines; PC3 and LNCaP. (A) The results of the MTT assay showing the viability of PC3 prostate cancer cells after HA treatment. Bright field photographs show morphological changes in PC3 (B) and LNCaP (C) cells treated with either 0.25, 0.5, and 1 µM of HA at 24 h. HA, holothurin A. Scale bar, 100 µM. Values represent mean + SD from triplicates. * p < 0.05; *** p < 0.001.
	Figure 3. Overexpression of RUNX1 in PC3 cell line. RUNX1 plasmids were transfected in human prostate cancer PC3 cell lines, which were observed by mRNA and protein expressions. The relative mRNA and protein expression levels of RUNX1 after transfection were detected by qRT-PCR (A) and Western blot (B). p < 0.01; * p < 0.001, compared with the vehicle control.
	Figure 4. HA suppresses mRNA and protein expressions of EMT-related proteins of PCa cell lines. (A) Following RUNX1 transfection, HA at 0.25, 0.5, and 1 µM were treated for 24 h. Protein expression levels and qRT-PCR of EMT markers were examined in RUNX1-overexpressing PC3 prostate cancer cells. qRT-PCR and Western blotting were observed in HA-treated PC3 (B) and LNCaP (C) cells. Values are expressed as mean ± SD. HA, holothurin A. * p < 0.05; p < 0.01; * p < 0.001, compared with the control. # p < 0.05; ## p < 0.01; ### p < 0.001, compared with the RUNX1-transfected group.
	Figure 5. HA suppresses RUNX1-mediated migration and invasion of PCa cell lines. Transwell assays were performed to assess cell migration and invasion in RUNX1-overexpressing PC3 cells (A) followed by a 0.25, 0.5, and 1 µM HA treatment and HA treatment alone in PC3 (B) and LNCaP (C) cells. Doc is used as a positive control. Magnification, ×10 and ×20. Values are expressed as the mean + SD. HA, holothurin A; Doc, docetaxel. * p < 0.05; p < 0.01; * p < 0.001, compared with the vehicle control.
	Figure 6. HA suppresses the RUNX 1-mediated enzymatic activity of MMPs of PCa cell lines. MMPs gelatinolytic activities were evaluated by gelatin zymography in RUNX1-overexpressing PC3 cells (A) followed by 0.25, 0.5, and 1 µM HA treatment and HA treatment alone in PC3 (B) and LNCaP (C) PCa cells. Doc is used as a positive control. HA, holothurin A; MMPs, matrix metalloproteinases; Doc, docetaxel. * p < 0.05; p < 0.01; * p < 0.001, compared with the control. # p < 0.05; ### p < 0.001, compared with the RUNX1-transfected group.
	Figure 7. HA inhibits EMT-mediated metastasis via inhibiting Akt, JNK, and P38 MAPK signaling pathways. PC3 cells were transfected with RUNX1 followed by HA treatment at 0.25, 0.5, and 1 µM of docetaxel for 24 h. Protein expression levels of important genes in the MAPK pathway were examined by a Western blot analysis (A). HA treatment alone is observed in PC3 (B) and LNCaP (C) PCa cells. Doc is used as a positive control. HA, holothurin A; Doc, docetaxel. * p < 0.05; p < 0.01; * p < 0.001, compared with the control. # p < 0.05; ## p < 0.01; ### p < 0.001, compared with the RUNX1-transfected group.

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	Figure 1. Chemical structure of holothurin A.
	Figure 2. The effect of HA on the viability of human PCa cell lines; PC3 and LNCaP. (A) The results of the MTT assay showing the viability of PC3 prostate cancer cells after HA treatment. Bright field photographs show morphological changes in PC3 (B) and LNCaP (C) cells treated with either 0.25, 0.5, and 1 µM of HA at 24 h. HA, holothurin A. Scale bar, 100 µM. Values represent mean + SD from triplicates. * p < 0.05; *** p < 0.001.
	Figure 3. Overexpression of RUNX1 in PC3 cell line. RUNX1 plasmids were transfected in human prostate cancer PC3 cell lines, which were observed by mRNA and protein expressions. The relative mRNA and protein expression levels of RUNX1 after transfection were detected by qRT-PCR (A) and Western blot (B). p < 0.01; * p < 0.001, compared with the vehicle control.
	Figure 4. HA suppresses mRNA and protein expressions of EMT-related proteins of PCa cell lines. (A) Following RUNX1 transfection, HA at 0.25, 0.5, and 1 µM were treated for 24 h. Protein expression levels and qRT-PCR of EMT markers were examined in RUNX1-overexpressing PC3 prostate cancer cells. qRT-PCR and Western blotting were observed in HA-treated PC3 (B) and LNCaP (C) cells. Values are expressed as mean ± SD. HA, holothurin A. * p < 0.05; p < 0.01; * p < 0.001, compared with the control. # p < 0.05; ## p < 0.01; ### p < 0.001, compared with the RUNX1-transfected group.
	Figure 5. HA suppresses RUNX1-mediated migration and invasion of PCa cell lines. Transwell assays were performed to assess cell migration and invasion in RUNX1-overexpressing PC3 cells (A) followed by a 0.25, 0.5, and 1 µM HA treatment and HA treatment alone in PC3 (B) and LNCaP (C) cells. Doc is used as a positive control. Magnification, ×10 and ×20. Values are expressed as the mean + SD. HA, holothurin A; Doc, docetaxel. * p < 0.05; p < 0.01; * p < 0.001, compared with the vehicle control.
	Figure 6. HA suppresses the RUNX 1-mediated enzymatic activity of MMPs of PCa cell lines. MMPs gelatinolytic activities were evaluated by gelatin zymography in RUNX1-overexpressing PC3 cells (A) followed by 0.25, 0.5, and 1 µM HA treatment and HA treatment alone in PC3 (B) and LNCaP (C) PCa cells. Doc is used as a positive control. HA, holothurin A; MMPs, matrix metalloproteinases; Doc, docetaxel. * p < 0.05; p < 0.01; * p < 0.001, compared with the control. # p < 0.05; ### p < 0.001, compared with the RUNX1-transfected group.
	Figure 7. HA inhibits EMT-mediated metastasis via inhibiting Akt, JNK, and P38 MAPK signaling pathways. PC3 cells were transfected with RUNX1 followed by HA treatment at 0.25, 0.5, and 1 µM of docetaxel for 24 h. Protein expression levels of important genes in the MAPK pathway were examined by a Western blot analysis (A). HA treatment alone is observed in PC3 (B) and LNCaP (C) PCa cells. Doc is used as a positive control. HA, holothurin A; Doc, docetaxel. * p < 0.05; p < 0.01; * p < 0.001, compared with the control. # p < 0.05; ## p < 0.01; ### p < 0.001, compared with the RUNX1-transfected group.