Aqueous Extract of Solanum nigrum Leaf Activates Autophagic Cell Death and Enhances Docetaxel-Induced Cytotoxicity in Human Endometrial Carcinoma Cells

Abstract

Chemotherapy is the main approach in dealing with advanced and recurrent endometrial cancer. An effective complementary ingredient can be helpful in improving the clinical outcome. Aqueous extract of Solanum nigrum leaf (AE-SN) is a principal ingredient for treating cancer patients in traditional Chinese medicinal practice but lacks sufficient evidence to verify its tumor suppression efficacy. This study evaluated the antitumor effects of AE-SN and also assessed the synergistic effects of AE-SN with docetaxel On the human endometrial cancer cell lines, HEC1A, HEC1B, and KLE. The activation of apoptotic markers, caspase-3 and poly-ADP-ribose polymerase, and autophagic marker, microtubule-associated protein 1 light chain 3 A/B, wAS determined to clarify the cell death pathways responsible for AE-SN induced tumor cell death. Results indicated that AE-SN-treatment has significant cytotoxicity on the tested endometrial cancer cells with accumulation of LC3 A/B II and demonstrated a synergistic effect of AE-SN and docetaxel in HEC1A and HEC1B cells, but not KLE cells. In conclusion, AE-SN treatment was effective in suppressing endometrial cancer cells via the autophagic pathway and was also capable of enhancing the cytotoxicity of docetaxel in human endometrial cancer cells. Our results provide meaningful evidence for integrative cancer therapy in the future.

Figure 1

Cytotoxicity of AE-SN in human endometrial cancer cells. (a–c) Results of MTT assay in HEC1A, HEC1B, and KLE cells. Cells were treated with 0, 0.05, 0.1, 0.2, 0.5, 1, 2 or 5 mg/mL AE-SN for 24, 48, or 72 hr. All three cell lines showed dose-dependent effects with AE-SN treatment at the three time points, as shown by one way ANOVA (P < 0.001). (d) Trypan blue exclusion test in HEC1A, HEC1B, and KLE cells. HEC1A and HEC1B cells were treated with 0, 0.1 or 0.5 mg/mL AE-SN, whereas KLE cells were treated with 0.5 and 2 mg/mL AE-SN for 48 hr. All three cell lines showed dose-dependent effects with AE-SN treatment, as shown by one-way ANOVA (P = 0.042, 0.043, and 0.001 for HEC1A, HEC1B, and KLE cells, resp.). Experiments were done in triplicate, and all data are presented as mean ± SD.

Figure 2

Morphological changes in AE-SN-treated human endometrial cancer cells. ((a) and (b)) HEC1A cells were treated with 0 or 0.5 mg/mL AE-SN for 48 hr. ((c) and (d)) HEC1B cells were treated with 0 or 0.5 mg/mL AE-SN for 48 hr. ((e) and (f)) KLE cells were treated with 0 or 0.5 mg/mL for 48 hr. Arrows indicate lipid droplet-like morphological changes in AE-SN-treated cells. (100x magnification).

Figure 3

Detection of cell death markers in AE-SN-treated human endometrial cancer cells. (a) HEC1A, HEC1B and KLE cells were treated with 0, 0.5, and 1.0 mg/mL AE-SN for 48 hr, and total protein extracts were harvested for western blotting analysis of the cell death markers, PARP, caspase-3 and LC3 A/B. (b) Accumulation of LC3 A/B II was semiquantified by Image J software. All experiments were replicated five times for statistical analysis of semiquantified data and are presented as mean ± SD. *Indicates statistical significance compared with 0 mg/mL AE-SN treatment by Student's t-test (P < 0.05).

Figure 4

Localization of LC3 A/B accumulation in AE-SN-treated HEC1A and HEC1B cells. ((a) and (b)) HEC1A cells were treated with 0 or 0.5 mg/mL AE-SN for 48 hr. ((c) and (d)) HEC1B cells were treated with 0 or 0.5 mg/mL AE-SN for 48 hr. Red, pseudocolorization on DAPI-stained cell nuclei. Green, LC3 A/B-stained cytoplasm (100x magnification).

Figure 5

Cytotoxicity of AE-SN and docetaxel cotreatment in human endometrial cancer cells. (a) HEC1A cells. (b) HEC1B cells. (c) KLE cells. Tested cells were treated with serial doses of docetaxel from 0 to 100 nM together with 0, 0.2, or 0.5 mg/mL AE-SN for 48 hr. Cell cytotoxicity was determined by MTT assay and presented as mean ± SD. Experiments were performed in triplicate.

Figure 6

Proposed mechanism for the programmed cell death activated by AE-SN and docetaxel in human endometrial cancer cells. + indicates synergistic effect of AE-SN and docetaxel.