ID09, A Newly-Designed Tubulin Inhibitor, Regulating the Proliferation, Migration, EMT Process and Apoptosis of Oral Squamous Cell Carcinoma

Abstract

Microtubules, a major target in oral squamous cell carcinoma (OSCC) chemotherapy, contribute to multiple malignant biological behaviors, including proliferation, migration, and epithelial-mesenchymal transition (EMT). Surpassing traditional tubulin inhibitors, ID09 emerges with brilliant solubility, photostability, and drug-sensitivity in multidrug-resistant cells. Its anti-tumor effects have been briefly verified in lung adenocarcinoma and hepatocellular carcinoma. However, whether OSCC is sensitive to ID09 and the potential mechanisms remain ambiguous, which are research purposes this study aimed to achieve. Various approaches were applied, including clone formation assay, flow cytometry, wound healing assay, Transwell assay, cell counting kit-8 assay, Western blot, qRT-PCR, and in vivo experiment. The experimental results revealed that ID09 not only contributed to cell cycle arrest, reduced migration, and reversed EMT, but accelerated mitochondria-initiated apoptosis. Remarkably, Western blot detected diminishment in expression of Mcl-1 due to the deactivation of Ras-Erk pathway, resulting in ID09-induced apoptosis, proliferation and migration suppression, which could be offset by Erk1/2 phosphorylation agonist Ro 67-7476. This study initially explored the essential role Mcl-1 played and the regulatory effect of Ras-Erk pathway in anti-cancer process triggered by tubulin inhibitor, broadening clinical horizon of tubulin inhibitors in oral squamous cell carcinoma chemotherapy application.

Keywords: Apoptosis; Malignant Biological Behaviors; Mcl-1; Oral Squamous Cell Carcinoma; Ras-Erk Pathway; Tubulin Inhibitor ID09.

Figure 1

Proliferation of SCC-15 and Cal-27 cells was suppressed by ID09. (A) Chemical structure of ID09. (B and C) Clone formation assay was performed after treating cells with different doses of ID09 (0, 10, 15, and 20 nM) or PTX (20 nM, positive control) for 24 hours. (D and E) Western blot detected PCNA expression level in SCC-15 and Cal-27 cells with indicated doses of ID09 (0, 10, 15, and 20 nM). (F and G) Flow cytometry was used to test the cell cycle after the cells were treated with ID09 (0, 10, 15, and 20 nM) or PTX (20 nM, positive control), revealing that ID09 could arrest cell cycle in the S and G₂/M phases. (H and I) Soft agar colony formation assay was effective in cell anchorage-independent growth ability measurement. The columns represent the means, and error bars represent standard deviations (NS, non-significant difference; */#P < 0.05; **/##P < 0.01; ***/### P < 0.001 versus the indicated group).

Figure 2

Effects of ID09 on the migration and invasion of SCC-15 and Cal-27 cells. (A and B) Wound healing rates in SCC-15 and Cal-27 cells after 24 and 48 hours were reduced by ID09 or PTX (positive control) in a dose-dependent manner. (C and D) Representative pictures of the Transwell assay performed in SCC-15 and Cal-27 cells treated with ID09 (0, 10, 15, and 20 nM) and PTX (20 nM, positive control) were exhibited, and the tendency of invasive cells was analyzed. (E) qRT-PCR was employed to measure the variation trend in mRNA expression levels of MMP-2 and MMP-9 in SCC-15 and Cal-27 cells treated with ID09 (0, 10, 15, and 20 nM) and PTX (20 nM, positive control). (F and G) Western blot was applied to measure the protein level of MMP-2 and MMP-9 in SCC-15 and Cal-27 cells treated with ID09 (0, 10, 15, and 20 nM). (H and I) In-gel zymography assay was selected to detect the activities of MMP2 and MMP9 in SCC-15 and Cal-27 cells. The columns represent the means, and error bars represent standard deviations (NS, non-significant difference; */#P < 0.05; **/##P < 0.01; ***/### P < 0.001 versus the indicated group).

Figure 3

ID09 influenced SCC-15 and Cal-27 cells morphology and EMT process through regulation of microtubules. (A) qRT-PCR was applied to detect the mRNA level variations of four kinds of tubulins, and (C) factors associated with epithelial-mesenchymal transformation were tested as well. (B) The cellular morphology changes induced by ID09 were photographed by microscope. (D and E) Western blot was used as a method to gauge β-catenin, E-Cadherin, N-Cadherin Vimentin, and α-tubulin expression levels. The columns represent the means and error bars represent standard deviations. (NS, non-significant difference; */#P < 0.05; **/##P < 0.01; ***/### P < 0.001 versus the indicated group).

Figure 4

Apoptosis via mitochondrial mechanism occured in SCC-15 and Cal-27 cells as a result of ID09 incubation. (A) CCK-8 Assay was used for detection to viability of SCC-15 and Cal-27 cells treated with different doses (0, 1, 2, 4, 8, 16, 32, 64, 128 nM) of ID09 for 24 hours or with 20 nM ID09 for different durations (0, 12, 24, 48, 60, 72 hours), PTX under same condition was used as a positive control. (B) Fluorescence pictures of OSCC cells marked by JC-1 probe were exhibited to detect mitochondrial membrane potential variation visually. (C and D) Mitochondrial membrane potential of SCC-15 and Cal-27 cells treated with ID09 were quantificationally measured via flow cytometry. (E and F) Flow cytometry was applied to test apoptosis degree of SCC-15 and Cal-27 cells incubated with ID09 or PTX (20 nM for positive control). (G and H) Western blot was utilized to measure the levels of apoptosis associated proteins extracted from cells incubated with ID09 (0 10 15 20 nM) for 24 hours, and the data were analyzed by Image J. The columns represent the means and error bars represent standard deviations. (NS, non-significant difference; */#P < 0.05; **/##P < 0.01; ***/### P < 0.001 versus the indicated group).

Figure 5

The effects of ID09 on Ras-Erk signaling pathway and Mcl-1. (A) qRT-PCR was utilized to detect the mRNA level variations of Bcl family factors. (B and C) Western blot was applied to verify the influence of ID09 treatment on Bcl family factors from protein level and (D and E) gauge the activation degree of Ras-Erk signaling pathway in treated SCC-15 and Cal-27 cells. The columns represent the means and error bars represent standard deviations. (NS, non-significant difference; */#P < 0.05; **/##P < 0.01; ***/### P < 0.001 versus the indicated group).

Figure 6

The proliferation inhibition, migration restraining and invasion suppression of SCC-15 and Cal-27 cells from ID09 incubation could be remedied by Ras pathway agonist Ro 67-7476. (A and B) Wound healing rates in SCC-15 and Cal-27 cells after 24 and 48 hours were reduced by ID09 but saved by Ro 67-7476. (C and D) The pictures of clone formation assay performed in OSCC cells incubated with ID09 and/or Ro67-7476 were displayed above. (E and F) Transwell assay was selected to measure the influence of ID09 and Ro 67-7476 on OSCC cells invasive ability. The columns represent the means and error bars represent standard deviations. (NS, non-significant difference; */#P < 0.05; **/##P < 0.01; ***/### P < 0.001 versus the indicated group).

Figure 7

Ras-Erk pathway agonist Ro 67-7476 is able to salvage ID09-mediated apoptosis in SCC-15 and Cal-27 cells. (A) Survival, apoptosis and necrosis in incubated OSCC cells were demonstrated visually via Hoechst 33342-PI double stain and (B and C) calculated mathematically by flow cytometry. (D and E) Western blot was utilized to analyze the variation of mitochondria apoptosis related factors inSCC-15 and Cal-27 cells. The columns represent the means and error bars represent standard deviations. (NS, non-significant difference; */#P < 0.05; **/##P < 0.01; ***/### P < 0.001 versus the indicated group).

Figure 8

Anti-tumor efficiency of ID09 on SCC-15 xenografts. (A) Picture of xenograft tumor was exhibited as follow. (B) HE staining was implemented to perform histological examination. (B, C and D) Average tumor volumes, tumor weights and body weights variation of each mice group were measured. (F and G) The expression of PCNA and Ki-67 in tumor tissue after ID09 treatment was visualized by IHC staining. (H) Cell apoptosis in tissue incubated by ID09 was detected by TUNEL staining. (I and J) Protein extracted from tumor tissue were determined by Western blot. The columns represent the means and error bars represent standard deviations. (NS, non-significant difference; */#P < 0.05; **/##P < 0.01; ***/### P < 0.001 versus the indicated group).

Figure 9

Anti-cancer effects of ID09 against various kinds of carcinomatous properties in OSCC and potential molecular mechanism. Binding to CBS, ID09 can fleetly catalyze microtubule depolymerization and cytoskeleton destruction, resulting in invasion and migration attenuation, EMT process reversion, and mitosis abnormality. In addition, due to deactivation of Ras-Erk pathway, the expression level of Mcl-1 is mitigated by ID09 treatment, resulting in triggering of caspase cascade and mitochondria-initiated apoptosis.