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 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 300µg/mL, 1700µg/mL, 0.5µM, 150µg/mL, 1800µg/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.

Effect of biotic and abiotic elicitors on production of betulin and betulinic acid in the hairy root culture of Betula pendula Roth.

Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem's inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150 mg l-1 on the 8th day. The highest content of BA was 1.3 mg g-1 DW after treatment with 1 mg l-1CCC on the 4th and 6th days and 200 mg l-1CTS NF on the 10th day. The highest B content (0.94 mg g-1DW) was obtained in the treated hairy root by 2 mg l-1 CCC after 4 and 6 days.

Improved accumulation of betulin and betulinic acid in cell suspension culture of Betula pendula roth by abiotic and biotic elicitors.

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5 mg L-1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200 mg L-1 of chitosan was found to be 5.9 × (6.5 mg g-1 DW) higher over control cells. Treating the cells with 2 mg L-1 of CCC, after 7 days, led to 149.3× enhancement of B content (19.4 mg g-1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation.