| Background: Tongue squamous cell carcinoma(TSCC) is the most common malignant oral cancer. TSCC is inclined to develop in the tongue edge, with early cervical lymph node metastasis. Due to the abundant lymphatic vessel and blood circulation as well as frequent movements of the tongue, the metastasis rate is relatively high. Even the comprehensive treatment including surgery, radiotherapy and chemotherapy has been introduced, the five-year survival rate of TSCC patients is relatively low. Chemotherapy is the critical therapy for late stage TSCC; however, it always generates chemoresistance which leads to the relapse of TSCC. Plumbagin is a kind of Chinese herbal medicine which is extracted from the root of Plumbago zeylanica L. Plumbagin possesses the anti-inflammatory, antibacterial, antioxidant and anticancer activities. However, the exact effect and relevant mechanisms of plumbagin on TSCC are not yet clear. Stable isotope labeling with amino acids in cell culture(SILAC) technology has the advantage of high marking efficience and accuracy, which is the gold standard of quantitative proteomics. SILAC can systematically explore the alterations of cellular proteins and reveal the regulation roles of related signaling pathways in tumor cells after the treatment of some certain drugs.Objectives: To explore the inhibition effect of plumbagin on TSCC cells as well as relevant molecular mechanisms. To further explore the chemosensitization and related machanisms of plumbagin on cisplatin in TSCC cells.Materials and Methods: 1. TSCC cell line SCC25 and CAL27 and normal gingival fibroblasts cell line HGF-1 have been used.2. MTT assay has been applied to examine the inhibition effect and half maximal inhibitory concentration(IC50) of plumbagin on SCC25 and CAL27 cells as well as HGF-1 cells. 3. MTT assay has been applied to examine the chemosensitization of plumbagin on cisplatin in TSCC cells. 4. SILAC proteomics has been employed to systematically reveal the alterations of cellular proteins and the regulation roles of related signaling pathways in TSCC cells after the treatment of plumbagin. 5. Colony formation assay has been applied to examine the inhibition effects of plumbagin and/or cisplatin in TSCC cells. 6. PI flow cytometry has been introduced to detect the distribution of cell cycle in TSCC cells after the treatment of plumbagin and/or cisplatin. 7. Annexin V: PE flow cytometry has been adopted to detect the apoptosis level in TSCC cells after the treatment of plumbagin and/or cisplatin. 8. Cyto ID flow cytometry has been adopted to detect the autophagy level in TSCC cells after the treatment of plumbagin and/or cisplatin. 9. Confocal microscopy system has been adopted to detect autophagy level in TSCC cells after the treatment of plumbagin. 10. CM-H2 DCFDA assay has been used to examine the cellular reactive oxygen species(ROS) level. 11. Western blotting has been used to examine the protein expressing level of epithelial to mesenchymal transition(EMT) and cancer stem cell(CSC) properties. 12. Western blotting has been used to examine the expressing level of the signaling pathway proteins.Results: 1. The alteration of cellular porteins and related signaling pathways after the treatment of plumbagin in TSCC cells:(1) After the treatment of plumbagin for 12, 24, 48, and 72 hours, the IC50 of SCC25 cells are 14.8, 8.4, 7.4, and 4.3 μM; the IC50 of CAL27 cells are 8.7, 3.1, 2.2, and 1.2 μM; the IC50 of HGF-1 cells are 35.7, 29.9, 17.4, and 8.1 μM.(2) The SILAC proteomics revealed that plumbagin can modulate 398 proteins and 101 signaling pathways in SCC25 cells; 354 proteins and 100 signaling pathways in CAL27 cells. 2. The role and mechanisms of plumbagin in cell cycle arrest, apoptosis and autophagy induction in TSCC cells:(1) Plumbagin can inhibit the colony formation of SCC25 and CAL27 cells via concentration/time dependent manner.(2) Plumbagin can induce G2/M arrest of SCC25 and CAL27 cells via concentration/time dependent manner. The expression level of cell cyle proteins cdc2, cyclin B1, and Cdc25 C were decreased, while the expression level of p53, p27, and p21 were increased.(3) Plumbagin can concentration/time dependently induce apoptosis of SCC25 and CAL27 cells. The expression level of Bcl-2 and Bcl-x L were decreased to different degrees, while the expression level of Bax, PUMA, Cytochrome C, FADD, TRADD, DR5, cleaved caspase-3, and cleaved caspase-9 were enhanced.(4) Plumbagin can concentration/time dependently induce autophagy of SCC25 and CAL27 cells. The expression level of autophagy protein Beclin 1 was increased as well as the ratio of LC3-II/LC3-I.(5) When the treatment of plumbagin was combined with SB202190 and Doxorubicin, respectively, the apoptosis and autophagy level were both enhanced. When the treatment of plumbagin was combined with Chloroquine and Z-VAD(OMe)-FMK, respectively, the apoptosis and autophagy level were both decreased. When the treatment of plumbagin was combined with EGFR, Anisomysin, and PA, respectively, the apoptosis and autophagy level were both declined.(6) The ratios of p-PI3K/PI3 K, p-Akt/Akt, p-m TOR/m TOR, and p-p38 MAPK/p38 MAPK were decreased at different levels when SCC25 and CAL27 were treated by plumbagin. 3. The role and mechanisms of plumbagin in EMT and CSC properties inhibition in TSCC cells:(1) Plumbagin can boost the generation of ROS in SCC25 and CAL27 cells and it can be blocked by ROS scavengers NAC and GSH.(2) The ratio of p-p38 MAPK/p38 MAPK and n-Nrf2/c-Nrf2 as well as the expression level of antioxidant response elements(ARE) NQO-1, GST, and HSP90 were declined at different degrees.(3) Plumbagin can inhibit EMT process in SCC25 and CAL27 cells with the elevation of E-cadherin/N-cadherin ratio.(4) Plumbagin can downregulate the expression level of CSC proteins Oct-4, Bmi-1, Nanog, and Sox-2 in SCC25 and CAL27 cells.(5) When the treatment of plumbagin was combined with NAC and GHS,respectively, the G2/M arrest, apoptosis and autophagy induction of SCC25 and CAL27 cells were all hampered as well as EMT and CSC inhibition. 4. The role and mechanisms of plumbagin in cisplatin chemosensitization:(1) When SCC25 cells were treated by cisplatin, the IC50 was 2.937 μM. When SCC25 cells were treated by the combination of cisplatin with 5 μM plumbagin, the IC50 was 0.634 μM; When CAL27 cells were treated by cisplatin, the IC50 was 23.33 μM. When CAL27 cells were treated by the combination of cisplatin with 1 μM plumbagin, the IC50 was 10.31 μM.(2) When SCC25 and CAL27 were treated by the combination of cisplatin with plumbagin, the colony formation was decreased at different levels comparing with cisplatin single group.(3) When SCC25 and CAL27 were treated by the combination of cisplatin with plumbagin, the percentage of G2/M phase cells were decreased at different levels comparing with cisplatin single group.(4) When SCC25 and CAL27 were treated by the combination of cisplatin with plumbagin, the percentage of apoptosis cells were decreased at different levels comparing with cisplatin single group.(5) When SCC25 and CAL27 were treated by the combination of cisplatin with plumbagin, the percentage of autophagy cells were decreased at different levels comparing with cisplatin single group.(6) When SCC25 and CAL27 were treated by the combination of cisplatin with plumbagin, the raitos of p-p38 MAPK/p38 MAPK, n-Nrf2/c-Nrf2, p-PI3K/PI3 K,p-Akt/Akt, and p-m TOR/m TOR were decreased at different levels comparing with cisplatin single group.Conclusions: 1. Plumbagin can efficiently inhibit the proliferation of TSCC cells, with low toxicology on normal gingival epithelial cells. Plumbagin can broadly regulate the cell cycle, apoptosis, autophagy, EMT and cellular redox status of TSCC cells. 2. Plumbagin can concentration/time dependently inhibit the colony formation and induce G2/M arrest in TSCC cells. Plumbagin can induce apoptosis and autophagy in TSCC cells via inhibition of PI3K/Akt/m TOR and p38 MAPK signaling pathways. There is a positive correlation between apoptosis and autophagy induced by plumbagin in TSCC cells. 3. Plumbagin can stimulate the generation of ROS via inhibition of p38 MAPK/Nrf2 signaling pathway. Plumbagin can remarkably inhibit EMT and CSC properties of TSCC cells. The ROS induced by plumbagin can participate in G2/M arrest, apoptosis and autophagy induction, EMT and CSC properties inhibition in TSCC cells. 4. Plumbagin can induce the G2/M arrest, apoptosis and autophagy by inhibiting PI3K/Akt/m TOR and p38 MAPK/Nrf2 signaling pathways, and then enhance the cisplatin chemosensitiviy in TSCC cells. |
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