Increased Induction of Apoptosis in ESCC (Esophageal Squamous-Cell Carcinoma) by Betula pendula Roth Stem Cell Extract containing Triterpenoids Compared to Doxorubicin

Background: Esophageal squamous-cell carcinoma (ESCC) is one of the most life-threatening malignancies worldwide, with a growing incidence in Iran higher than the global average.

Objective: The present study, for the first time under patent number (97668), introduces a method using in vitro production of activated-Birch stem cells using biotechnological techniques of tissue culture and plant stem cell culture from Betula pendula Roth (Birch) bark.

Methods: In the first step, Birch stem cells were produced in large amounts using tissue culture, and then the amount of triterpenoids of its extract was measured by the HPLC method. In the second step, the cytotoxicity was evaluated by MTT, and the IC50 was calculated. The cellular apoptosis in response to the extract compared to doxorubicin was measured using the Annexin V kit and the flow cytometry method.

Results: The optimized method introduced in the current study efficiently produced plant stem cells containing triterpenoids in large quantities over a period of 2-4 months. Our findings indicated that the growth of ESCC cells decreased by induction treatment at 3 times (24, 36, 48 hours). IC50 values were obtained in 24 hours for the natural bark extract, Birch stem cell extract, doxorubicin and interactions of two extracts with doxorubicin at 300 µg/mL, 1700 µg/mL, 0.5 µM, 150 µg/mL, 1800 µg/mL, respectively. In the flow cytometric test, the Birch stem cell extract showed the highest percentage of apoptosis with 92.5% for total apoptosis. The percentage of total apoptosis in doxorubicin treatment was 85.33%, and the combination of doxorubicin with Birch stem cell extract was 88.33%. Natural bark extract and its combination with a lower percentage (69.33% and 70.33%, respectively) caused apoptosis of esophageal cancer cells.

Conclusion: Owing to the extinction of Birch in Iran and its inaccessibility and exploitation, Birch stem cells can be cultured as an appropriate alternative source to produce valuable triterpenoids for pharmaceutical purposes. Additionally, according to the results of this study, stem cells can be used to enhance the treatment of esophageal cancer and supplementation with chemotherapy.

Keywords: Apoptosis; ESCC cancer; birch; doxorubicin.; plant stem cells; triterpenoids.