Cell experiment:

The MTT assay is used to evaluate the overall functional integrity and viability of the cultured cells. The MCF-7 cells are put into 96-well plates (2×104 for each well), which are incubated for 24 h. The specific concentrations of alpha-Amanitin and β-Amanitin are added to the cell culture medium, and plates are incubated for an additional 36 h. MTT solution (1:10 ratio) and dimethyl sulfoxide (DMSO) (100 μL) are then added to the cell culture medium and plates are incubated overnight. The absorbance is measured at 570 nm on a plate reader. This experiment is repeated 3 times. The absorbance data are calculated as percentages according to the control group.

Animal experiment:

For tumorigenicity tests, six colonies (untreated) and DTCs derived from MKN45 cells are individually injected subcutaneously into the left and right side of the backs of six 6-week-old female nude mice (BALB/cAjcl-nu/nu). These mice are monitored for 49 days after the inoculation or until tumors reach 10 mm in the largest diameter, and are then euthanized. For the PC model, 1.0×106 MKN45 cells are injected intraperitoneally into six 6-week-old female nude mice (BALB/cAjcl-nu/nu). Mice are then treated with CIS (4.0 mg/kg, intraperitoneal administration) or a combination of CIS and alpha-Amanitin (0.4 mg/kg, intraperitoneal administration). For the combination treatment, alpha-Amanitin is given 24 hours before CIS. Body weight is monitored for 28 days after the treatment.

alpha-Amanitin is the principal toxin of several deadly poisonous mushrooms, exerting its toxic function by inhibiting RNA-polymerase II.

Alpha-Amanitin decreases TAF15 mRNA and TAF15 protein levels in MKN45 cells, and inhibits the RNAPII activity towards TAF15 mRNA[2]. alpha-Amanitin decreases cell viability by 14%, 21%, 41%, 44%, and 50% at concentrations of 100, 10, 1, 0.1, and 0.01 μg/mL, respectively. The LD50 of the alpha-Amanitin at 36 h is measured as 1 μg/mL. The total amount of protein within the cell at 24 h is significantly increased for the 1 μg/mL dose of alpha-Amanitin compared to the control[3]. Alpha-Amanitin dramatically decreases the expression of gap junctional genes (Gja1, Gja4 and Gjc1) and FSHr and LHr in cumulus cells[4].

The intravenous LD50 dose of alpha-Amanitin is 0.327 mg/kg body weight after intravenous injection into BALB/c mice. After 12 h of alpha-Amanitin injection in caudal vein, the levels of WBC, RBC and HGB decrease significantly, while those of BUN and Crea increase significantly in serum. alpha-Amanitin inhibits some genes (Hsp90b1, Irx4, etc.), whose encoded proteins regulate the RNA polymerase II activity. alpha-Amanitin down-regulates some proteins (Nmi, Trpc5, etc.) taking part in the transcription progress[1]. alpha-Amanitin has potent activity in DTC suppression. Mice injected with alpha-Amanitin (0.4 mg/kg, i.p.)-treated cells maintain their body weight, while those receiving a peritoneal injection of MKN45 cells show a constant decrease in body weight[2].

References:
[1]. Zhao J, et al. Pathological effects of the mushroom toxin alpha-amanitin on BALB/c mice. Peptides. 2006 Dec;27(12):3047-3052.
[2]. Kume K, et al. α-Amanitin Restrains Cancer Relapse from Drug-Tolerant Cell Subpopulations via TAF15. Sci Rep. 2016 May 16;6:25895
[3]. Kaya E, et al. Evaluation and comparison of alpha- and beta-amanitin toxicity on MCF-7 cell line. Turk J Med Sci. 2014;44(5):728-32
[4]. Park MW, et al. RNA Polymerase II Inhibitor, α-Amanitin, Affects Gene Expression for Gap Junctions and Metabolic Capabilities of Cumulus Cells, but Not Oocyte, during in vitro Mouse Oocyte Maturation. Dev Reprod. 2013 Mar;17(1):63-72