Fig. 3. Gimatecan reduces topoisomerase I specific activity and suppress topoisomerase I expression in ESCC.

a, b Experiment of topoisomerase I activity using different assay: Eca-109 and KYSE-450 cell lines were exposed to serial dilutions (10 nM, 20 nM, and 40 nM) of gimatecan and irinotecan for 2 h, and 20 µL of reaction containing nucleoli extract protein was incubated with supercoiled DNA for 30 min. Lane 1, supercoiled DNA only. Lane 2, relaxed DNA used as negative control. Lanes 3–5, supercoiled DNA, and nucleoli extract protein of cells treated with different concentration of gimatecan or irinotecan for 2 h; c, d the expression of topoisomerase 1 was assessed by Western blotting in vitro and in vivo. Eca-109 and KYSE-450 cell lines were exposed to 10 nM, 20 nM, and 40 nM gimatecan or irinotecan for 48 h, and harvested at 70–80% confluence. For in vivo experiment, the mice were killed and tumor tissues of Eca-109 and KYSE-450 cell line xenografts and PDX models were harvested at the end of treatment. Total protein was extracted from harvested cell lines or tumor tissues, and the expression of topoisomerase 1 was assessed by Western blotting