The combination of alisertib, an investigational Aurora kinase A inhibitor, and docetaxel promotes cell death and reduces tumor growth in preclinical cell models of upper gastrointestinal adenocarcinomas

Abstract

Background: Upper gastrointestinal adenocarcinomas (UGCs) respond poorly to current chemotherapeutic regimes. The authors and others have previously reported frequent Aurora kinase A (AURKA) gene amplification and mRNA and protein overexpression in UGCs. The objective of the current study was to determine the therapeutic potential of alisertib (MLN8237) alone and in combination with docetaxel in UGCs.

Methods: After treatment with alisertib and/or docetaxel, clonogenic cell survival, cell cycle analyses, Western blot analyses, and tumor xenograft growth assays were carried out to measure cell survival, cell cycle progression, apoptotic protein expression, and tumor xenograft volumes, respectively.

Results: By using the AGS, FLO-1, and OE33 UGC cell lines, which have constitutive AURKA overexpression and variable tumor protein 53 (p53) status, significantly enhanced inhibition of cancer cell survival was observed with alisertib and docetaxel treatment in combination (P < .001), compared with single-agent treatments. Cell cycle analyses, after 48 hours of treatment with alisertib, produced a significant increase in the percentage of polyploidy in UGC cells (P < .01) that was further enhanced by docetaxel (P < .001). In addition, an increase in the percentage of cells in sub-G1-phase observed with alisertib (P < .01) was significantly enhanced with the combination treatment (P < .001). Western blot analysis demonstrated higher induction of cleaved caspase 3 protein expression with the combined treatment compared with single-agent treatments. In addition, FLO-1 and OE33 cell xenograft models demonstrated enhanced antitumor activity for the alisertib and docetaxel combination compared with single-agent treatments (P < .001).

Conclusions: The current study demonstrated that alisertib combined with docetaxel can mediate a better therapeutic outcome in UGC cell lines.

Figure 1. Alisertib/MLN8237 and Docetaxel combination treatment significantly inhibits cell survival

The cell survival assay data indicates significant inhibition of AGS (A), FLO-1 (B) and OE33 (C) cell survival after treatment with Alisertib (MLN) and Docetaxel combination. The AGS, FLO-1 and OE33 cells were treated with Alisertib (0.5μM) and/or Docetaxel (0.5nM, 1.0nM & 5.0nM) for 24hr and incubated in drug free medium for 10 days. Alisertib (0.5μM) and Docetaxel (1.0nM) combination treatment significantly suppressed survival of cells. CV - Control Vehicle; MLN 0.5 - MLN8237/Alisertib 0.5μM; DOCE 0.5 - Docetaxel 0.5nM; DOCE 1.0 - Docetaxel 1.0nM, DOCE 5.0 - Docetaxel 5.0nM,* p<0.05 and ** p<0.01.

Figure 2. Alisertib/MLN8237 and Docetaxel combination treatment enhances polyploidy and alters cell cycle progression

AGS cells were treated with Alisertib (0.1μM) and/or Docetaxel (0.5nM) for 24hr and 48hr, respectively, and cell cycle progression was analyzed with flow cytometry. After 24hr (A) and 48hr (B) of treatment, Alisertib (0.1μM) in combination with Docetaxel (0.5nM) significantly enhanced polyploidy in AGS cells. CV - Control Vehicle; MLN - MLN8237/Alisertib; DOCE - Docetaxel, * p<0.05 and ** p<0.01.

Figure 3. Alisertib/MLN8237 and Docetaxel combination treatment enhances polyploidy and alters cell cycle progression

A–B) FLO-1 cells were treated with Alisertib (0.1μM) and/or Docetaxel (0.5nM) in cell culture medium (2.5% FBS) for 24hr and 48hr, respectively, and cell cycle progression was analyzed with flow cytometry. (A) After 24hr of treatment, Alisertib in combination with Docetaxel induces G2-M-phase arrest, suppresses G1-phase and enhances apoptosis (sub-G1) in FLO-1 UGC cells. (B) After 48hr of treatment, Alisertib and Docetaxel combination treatment significantly increases the percentage of sub-G1-phase cells in FLO-1 UGC cells. C–D) OE33 cells were treated with Alisertib and/or Docetaxel for 24hr and 48hr, respectively, and cell cycle progression was analyzed with flow cytometry. (C) After 24hr of treatment, Alisertib in combination with Docetaxel induces G2-M-phase arrest and suppresses G1 and S-phases, respectively in OE33 UGC cells. (D) After 48hr of treatment, Alisertib and Docetaxel in combination enhances polyploidy in OE33 cells. CV - Control Vehicle; MLN -MLN8237/Alisertib; DOCE - Docetaxel, * p<0.05 and ** p<0.01.

Figure 4. Alisertib/MLN8237 and Docetaxel combination treatment significantly enhances apoptotic marker expression

The AGS (A), FLO-1 (B), and OE33 (C) cells were treated with Alisertib and/or Docetaxel for 48hr. Alisertib and Docetaxel combination treatment significantly enhanced expression of cleaved caspase 3 in AGS, FLO-1 and OE33 UGC cells. CV - Control Vehicle; MLN - MLN8237/Alisertib; DOCE – Docetaxel.

Figure 5. Alisertib/MLN8237 and Docetaxel combination treatment exhibits enhanced anti-tumor activity in vivo

FLO-1 and OE33 tumor xenografts were treated with Alisertib (30mg/kg) and/or Docetaxel (10mg/kg) for 21 days and tumor size was measured every alternate day. (A & B) The data indicates that MLN8237 (30mg/kg) and Docetaxel (10mg/kg) combination treatment has significantly enhanced anti-tumor activity against FLO-1 and OE33 tumor xenografts. MLN - MLN8237/Alisertib; DOCE – Docetaxel, * p<0.05 and ** p<0.01.

Figure 6. Alisertib/MLN8237 and Docetaxel combination treatment suppresses proliferation and enhances apoptotic marker expression in FLO-1 tumor xenografts

FLO-1 tumor xenografts were treated with Alisertib (30mg/kg) and/or Docetaxel (10mg/kg) for 21 days. Subsequently, tumors were isolated and immunohistochemical analyses were done to measure Ki-67 and cleaved caspase 3 expression. (A) The data indicates that Alisertib (30mg/kg) and Docetaxel (10mg/kg) combination treatment significantly inhibits Ki-67 expression in FLO-1 tumor xenografts. (B) Combination treatment with Alisertib (30mg/kg) and Docetaxel (10mg/kg) exhibits enhanced cleaved caspase 3 protein expression in FLO-1 tumor xenografts. MLN - MLN8237/Alisertib; DOCE – Docetaxel, * p<0.05 and ** p<0.01.