1. ? SUMMARY INTRODUCTION RESULTS CONCLUSIONS
CHARACTERIZATION OF A Caesalpinia spinosa EXTRACT THAT CONTROLS BREAST CANCER STEM CELLS BY OVERCOMING DETOXIFYING MECHANISM AND BY TRIGGER IMMUNOGENIC MOLECULE CALRETICULIN
Tito A. Sandoval1, Claudia P. Urueña1, Mónica Llano1, Alejandra Gomez, John F. Hernandez1, Alfonso Barreto and Susana Fiorentino1*
Grupo de Inmunobiología y Biología Celular, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia.
SUMMARY
An anti-tumor extract of C. spinosa (P2Et) previously obtained by our group, exhibits cytotoxic activity on tumor cell lines and reduce primary breast and melanoma tumor in BALB/c and C57BL/6 mice. Interestingly, P2Et also reduce lung and spleen metastasis from breast cancer cells in mice, due to activation of specific immune response against the tumor1-2. However, another possibility is a direct activity against Cancer stem cells (CSC), the primary cell type responsible for tumor metastasis and relapse, which have the ability to growth forming multicellular spheroids and express ABC-transporters that confers resistance to chemotherapeutic agents and detoxifying molecules as ALDH. In this study, we thoroughly characterized the P2Et extract by using HPLC/MS and isolated two main compounds, gallic acid and ethyl gallate. In addition, we evaluated P2Et cytotoxic activity in breast CSC, alone and in combination with doxorubicin. Finally, we evaluated the expression of the immunogenic cell dead molecule Calreticulin. Our results together suggest that P2Et might be a promising adjuvant for treating drug-resistant tumors acting on resistant and metastatic cancer stem cells
INTRODUCTION
EVALUATION OF THE EFFECT OF P2Et AGAINST CSC
A. Cytotoxicity
C. Mammosphere inhibition ?
B. Synergy with Dox
RESULTS
CHEMICAL CHARACTERIZATION (HPLC/MS)
A. Cytotoxicity of P2Et in CSC models (IC50) B. Synergy of P2Et with doxorubicin +++ Synergy, ++ moderate synergy, ± additivity. C. Mammosphere inhibition in CSC models (SFE: Sphere Formation Efficiency)
in vivo SYNERGY WITH DOX and in vitro CALRETICULIN
Gallic acid derivatives
Identificated compounds: 1. n-O-galloylquinic (tR: min); 2. gallic acid (tR=5.64); 3. di-O-galloylquinic acid (tR: ); 4. galloylquinic acid ethyl ester (tR: ); 5. tri-O-galloylquinic acid (tR: min); 6. tetra-O-galloylquinic acid (tR: min); 7. tri-O-galloyquinic acid (tR: min); 8. ethyl gallate (tR =22.70). Other compounds: *unknown tetra-GCQ; tri-O-galloyl quinic acid ethyl ester; *di-O-galloyl quinic acid ethyl gallate ester; tri-O-galloyl quinic acid ethyl ester; *tri-O-galloyl quinic acid ethyl gallate ester. * tentative identification.
CSC MODEL DEVELOPMENT AND CHARACTERIZATION
4T1 H17 (ALDH+) A.
A. Tumor development and survival B
MES/SA Dx5
4T1
4T1 H17
TS/A
Flow cytometry
B. Calreticulin
A. Tumor development after treatment with P2Et, Doxorubicin and its combination *** p< * p<0.05 ns: non-significant B. Calreticulin expression after treatment with P2Et, Doxorubicin and its combination. C.
CONCLUSIONS
P2Et controls breast cancer stem cells by overcoming detoxifying mechanism ALDH and drug-efflux mechanism, in addition exhibited synergistic effect with doxorubicin in vitro and in vivo. Calreticulin overexpression after treatment alone and in combination with DOX shows that immune response its important for anti-tumor activity. These results together suggest that P2Et might be a promising adjuvant for treating drug-resistant tumors acting on resistant and metastatic cancer stem cells. We are currently obtaining the P2Et extract in GMP conditions, in order to conduct a phase-I clinical trial in patients with breast cancer attending the Javeriana Oncology Center at the San Ignacio Hospital in Bogota.
A. 4T1 H17 model was stablished from 4T1 parental line by serial passages of lung metastasis recovered cells. B. Cells were characterized by flow cytometry with cancer stem cells markers C. Drug Efflux and CSC markers expression
ACKNOWLEDGEMENTS
The authors thank Pontificia Universidad Javeriana for their support and Departamento Administrativo de Ciencia, Tecnología e Innovación COLCIENCIAS (grant number ) Bogotá, Colombia, for financial support and to Colombian Environmental Ministry for allowing use of genetic resources and derived products (agreement number 0454 of 15/05/2013).