Effective Management of Advanced Angiosarcoma by the Synergistic Combination of Propranolol and Vinblastine-based Metronomic Chemotherapy: A Bench to Bedside Study

Abstract

Background: Angiosarcomas are rare malignant tumors of vascular origin that represent a genuine therapeutic challenge. Recently, the combination of metronomic chemotherapy and drug repositioning has been proposed as an attractive alternative for cancer patients living in developing countries.

Methods: In vitro experiments with transformed endothelial cells were used to identify synergistic interactions between anti-hypertensive drug propranolol and chemotherapeutics. This led to the design of a pilot treatment protocol combining oral propranolol and metronomic chemotherapy. Seven consecutive patients with advanced/metastatic/recurrent angiosarcoma were treated with this combination for up to 12months, followed by propranolol-containing maintenance therapy.

Findings: Gene expression analysis showed expression of ADRB1 and ADRB2 adrenergic receptor genes in transformed endothelial cells and in angiosarcoma tumors. Propranolol strongly synergized with the microtubule-targeting agent vinblastine in vitro, but only displayed additivity or slight antagonism with paclitaxel and doxorubicin. A combination treatment using bi-daily propranolol (40mg) and weekly metronomic vinblastine (6mg/m(2)) and methotrexate (35mg/m(2)) was designed and used in 7 patients with advanced angiosarcoma. Treatment was well tolerated and resulted in 100% response rate, including 1 complete response and 3 very good partial responses, based on RECIST criteria. Median progression-free and overall survival was 11months (range 5-24) and 16months (range 10-30), respectively.

Interpretation: Our results provide a strong rationale for the combination of β-blockers and vinblastine-based metronomic chemotherapy for the treatment of advanced angiosarcoma. Furthermore, our study highlights the potential of drug repositioning in combination with metronomic chemotherapy in low- and middle-income country setting.

Funding: This study was funded by institutional and philanthropic grants.

Keywords: Adrenergic receptor; Angiosarcoma; Metronomic chemotherapy; Propranolol; Vascular tumor.

Fig. 1

Expression of adrenergic receptor genes in immortalized and Ras-transformed vascular endothelial cells and in vitro sensitivity to propranolol. (A) Relative mRNA expression of ADRB1 and ADRB2 adrenergic receptor genes in immortalized (HMEC-1, BMhTERT-1 and BMST) and Ras-transformed (BMST-Ras) endothelial cell lines as determined by qRT-PCR using GAPDH as control gene. SK-N-MC neuroepithelioma cell line and HeLa cervical cancer cell line were included as positive controls for ADRB1 and ADRB2 expression, respectively. (B) Growth inhibition assay performed on BMST (black) and BMST-Ras (red) cell lines using Alamar Blue after 72 h incubation with propranolol. Points, % of cell proliferation as compared to untreated control cells, means of eight individual experiments; bars, 95% confidence interval; log scale for x axis.

Fig. 2

In vitro drug combination studies. Growth inhibition assays performed on BMST (left panel) and BMST-Ras (right panel) cell lines using Alamar Blue after 72 h incubation with doxorubicin (A), paclitaxel (B) and vinblastine (C) alone (black — solid line) or in combination with propranolol at 1 μM (black — broken line), 10 μM (green — solid line) and 50 μM (red — solid line). Points, % of cell proliferation as compared to untreated control cells, means of four individual experiments; bars, 95% confidence interval; log scale for x axis. Statistical analysis was performed by comparing the cytotoxic effect of chemotherapy alone and in combination with propranolol using Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig. 3

Changes in sensitivity to chemotherapy. Histogram representation of the molar concentration of doxorubicin (A), paclitaxel (B) and vinblastine (C) required to inhibit 50% (IC₅₀) of cell proliferation after 72 h drug incubation in absence (black) or presence of propranolol at 1 μM (hashed), 10 μM (green) and 50 μM (red). Columns, means of four individual experiments; bars, SEM. Statistical analysis was performed by comparing the IC₅₀ values of chemotherapy alone and in combination with propranolol using Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig. 4

Combination indexes of propranolol with chemotherapy agents. Dot plot representation of the combination index of propranolol in association with doxorubicin, paclitaxel and vinblastine on BMST (□) and BMST-Ras (○) cell lines. Growth inhibition assays were performed using Alamar Blue after 72 h incubation with a range of chemotherapeutic drug concentrations in the presence or absence of propranolol at 50 μM. CI values were determined based on the Chou and Talalay method for all tested concentrations of chemotherapeutic drug. Bars, mean of at least three individual experiments; y axis, log scale.

Fig. 5

Combination of propranolol and vinblastine in tumor spheroids. (A) Quantitative analysis of the growth of tumor spheroids formed by BMST-Ras cells on ultra-low attachment plates by daily volume measurements. Tumor spheroids were either untreated (black) or treated with 10 μM propranolol (green), 1 nM vinblastine (blue) or the combination (red). Points, means of at least three individual experiments; bars, SEM. Statistical analysis was performed by comparing the volume of tumor spheroids in absence and presence of treatment using Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (B) Representative photographs of BMST-Ras tumor spheroids after 120 h incubation with vinblastine and propranolol alone or in combination. Images were obtained using the 5 × objective of a Zeiss Axiovert 200 M. Inset, % of growth inhibition as compared to untreated tumor spheroids; scale bar, 200 μm. (C) Representative immunoblots of BMST-Ras tumor spheroid lysates after 120 h incubation with no drug (1), 10 μM propranolol (2), 1 nM vinblastine (3) or the combination (4). Membranes were probed with antibodies directed against cleaved PARP and GAPDH (loading control).

Fig. 6

Expression of β-adrenergic receptor gene in angiosarcoma tumors. ADRB1 (top panel — 103 base pairs) and ADRB2 (bottom panel — 138 base pairs) RT-PCR products analyzed by 2% agarose gel electrophoresis followed by ethidium bromide staining using 50 bp DNA marker. mRNA was isolated from tumor material obtained from patients #1, #3 and #4. Positive control RNA was extracted from HeLa (ADRB1) and SK-N-SH (ADRB2) cell lines.

Fig. 7

Clinical response of angiosarcoma patients. (A) Photographs of patient #2 who presented with a large angiosarcoma in the periorbital region and multiple lesions on the scalp. Sustained complete clinical response was observed in this patient. (B–C) PET and PET–CT scan images of patient #3 who presented with a primary angiosarcoma of the scalp and multiple metastases located in the vertebrae (arrows). Sustained complete clinical and metabolic response was observed in this patient.