TRAIL and doxorubicin combination induces proapoptotic and antiangiogenic effects in soft tissue sarcoma in vivo

Abstract

Purpose: Novel therapeutic approaches for complex karyotype soft tissue sarcoma (STS) are crucially needed. Consequently, we assessed the efficacy of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in combination with chemotherapy, on local and metastatic growth of human STS xenografts in vivo.

Experimental design: TRAIL was evaluated alone and combined with low-dose doxorubicin in two human STS severe combined immunodeficient mouse xenograft models using fibrosarcoma (HT1080; wild-type p53) and leiomyosarcoma (SKLMS1; mutated p53), testing for effects on local growth, metastasis, and overall survival. Magnetic resonance imaging was used to evaluate local growth and bioluminescence was used to longitudinally assess lung metastases. Tissues were evaluated through immunohistocemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining for treatment effects on tumor cell proliferation, apoptosis, angiogenesis, angiogenic factors, and TRAIL receptor expression. Quantitative real-time polymerase chain reaction (QRTPCR) angiogenesis array was used to assess therapy-induced gene expression changes.

Results: TRAIL/doxorubicin combination induced marked STS local and metastatic growth inhibition in a p53-independent manner. Significantly increased (P < 0.001) host survival was also demonstrable. Combined therapy induced significant apoptosis, decreased tumor cell proliferation, and increased TRAIL receptor (DR4 and DR5) expression in all treated tumors. Moreover, decreased microvessel density was observed, possibly secondary to increased expression of the antiangiogenic factor CXCL10 and decreased proangiogenic interleukin-8 cytokine in response to TRAIL/doxorubicin combination, as was also observed in vitro.

Conclusions: Given the urgent need for better systemic approaches to STS, clinical trials evaluating TRAIL in combination with low-dose chemotherapy are potentially warranted.

Figure 1

Doxorubicin (Dox) and TRAIL combination results in superior pro-apoptotic effects in STS cells in vitro. A) The combination of doxorubicin (0.1μM/48hr) and TRAIL (25μg/ml/24hr) exhibits superior STS growth inhibition effects that either compound alone (MTS assays; *p<0.005); B) The combination of doxorubicin (0.1μM/48hr) and TRAIL (25μg/ml/24hr) elicits significant apoptosis in STS cells (PI/FACS analysis; *p<0.05); C) Pro-apoptotic effect is also demonstrated via caspase-3 activity assay (*p<0.05). Graphs represent the average of three repeated experiments ±SD.

Figure 2

Doxorubicin (1.2mg/kg/biweekly) followed by TRAIL (10mg/kg, five days per week) decreases leiomyosarcoma (SKLMS1) growth in vivo. A) Tumor growth curves per treatment group (10mice/group); B) Representative MRI images of STS xenografts at time of treatment initiation and by the end of the study. Graphs depict average tumor volume as calculated from MRI images (n=3/group; *p<0.05). White bars represent tumor volume at treatment initiation and black bars at study termination; C) PCNA immunohistochemistry and TUNEL staining (red = nuclei, green =TUNEL) of SKLMS1 xenograft specimens (selected for presence of viable tumor shown in H+E); and, D) DR4 (green) and DR5 (red) immunoflorescent staining of SKLMS1 xenografts (nuclei are depicted in blue).

Figure 3

Doxorubicin and TRAIL combination decreases fibrosarcoma (HT1080) growth in vivo. A) Tumor growth curves per treatment group (left; n=10mice/group, p<0.01) and tumor pictures (right upper panel). Box plots (right lower panel) depict average tumor weights at termination of the study (p<0.001); B) Ki-67 immunoflorescence (blue =nuclei, green = Ki-67) and TUNEL staining (blue = nuclei, green =TUNEL) of HT1080 xenograft specimens (selected for presence of viable tumor shown in H+E); C) DR4 (green) and DR5 (red) immunoflorescent staining of HT1080 xenografts (nuclei are depicted in blue).

Fig 4

Doxorubicin and TRAIL combination inhibits fibrosarcoma (HT1080) lung metastases growth and enhances survival. A) BLI images of one representative mice of each treatment arm are shown demonstrating the markedly decreased luciferase readout in combination treated mice compared to control or either compound alone. Pictures and H+E staining demonstrate the reduction in lung metastatic deposits (arrows) in combination treated mice; B) Box plots demonstrating the significant decrease in lung weight of combination treated mice (*p<0.005); C) Kaplan Meier survival curves demonstrating a statistically significant prolongation in overall survival of mice treated with doxorubicin/TRAIL combination (p=0.001)

Fig 5

Doxorubicin and TRAIL combination inhibits STS associated angiogenesis. A) CD31 immunohistochemisty and CD31/TUNEL double immunoflorescence (CD31 = red, TUNEL = green) demonstrate decrease in blood vessel density in combination treatment samples but no evidence of endothelial cell apoptosis; B) Angiogenesis RT PCR array demonstrated a significant increase in CXCL10 mRNA expression and a marked decrease in IL8 mRNA levels in combination treated tumor samples of both STS xenograft models; C) Effects of treatment on CXCL10 and IL8 mRNA expression was further confirmed via RT PCR using an independent cohort of tumor samples (*p<0.05). No significant gene expression changes were noticed in samples treated with either agent alone; D) effect of treatment on IL8 (red) and CXCL10 (red) protein expression in vivo was evaluated by immunoflorescence (nuclei = blue). An increase in tumor infiltrating macrophages and a decrease in tumor infiltrating neutrophils was also observed in TRAIL/doxorubicin treated tumors. Graphs represent the average of three repeated experiments ±SD.

Fig 6

Doxorubicin and TRAIL combination modulates angiogenesis-related factor expression in STS cells. A) STS cells grown in culture were treated with doxorubicin (0.1μM/24hr), TRAIL (25μg/ml/12hr), or their combination. qRTPCR demonstrated a significant increase in CXCL10 mRNA levels (*p<0.05) and a decrease in IL-8 mRNA expression (*p<0.05); B) Conditioned media was collected from STS cells treated as above and was subjected to ELISA. A significant increase in CXCL10 protein expression (*p<0.05) and a decrease in IL8 protein levels (*p<0.05) were observed. Graphs represent the average of three repeated experiments ±SD.