Aqueous Extract of Solanum nigrum Leaves Induces Autophagy and Enhances Cytotoxicity of Cisplatin, Doxorubicin, Docetaxel, and 5-Fluorouracil in Human Colorectal Carcinoma Cells

Abstract

Colorectal cancer is a common cancer worldwide, and chemotherapy is a mainstream approach for advanced and recurrent cases. Development of effective complementary drugs could help improve tumor suppression efficiency and control adverse effects from chemotherapy. The aqueous extract of Solanum nigrum leaves (AE-SN) is an essential component in many traditional Chinese medicine formulas for treating cancer, but there is a lack of evidence verifying its tumor suppression efficacy in colorectal cancer. The purpose of this study is to evaluate the tumor suppression efficacy of AE-SN using DLD-1 and HT-29 human colorectal carcinoma cells and examine the combined drug effect when combined with the chemotherapeutic drugs cisplatin, doxorubicin, docetaxel, and 5-fluorouracil. The results indicated that AE-SN induced autophagy via microtubule-associated protein 1 light chain 3 A/B II accumulation but not caspase-3-dependent apoptosis in both cell lines. The IC50s after 48 hours of treatment were 0.541 and 0.948 mg/ml AE-SN in DLD-1 and HT-29, respectively. AE-SN also demonstrated a combined drug effect with all tested drugs by enhancing cytotoxicity in tumor cells. Our results suggest that AE-SN has potential in the development of complementary chemotherapy for colorectal cancer.

Figure 1

Cytotoxicity of AE-SN in DLD-1 and HT-29 human colorectal carcinoma cells (a); DLD-1 cells were treated with 0.05 to 5 mg/mL AE-SN for 24 or 48 hr; (b), HT-29 cells were treated with 0.05 to 5 mg/mL AE-SN for 24 hr; the cytotoxicity of (a) and (b) was analyzed by MTT assay; (c)DLD-1 or HT-29 cells were treated with 0.2 to 1 mg/mL AE-SN; and the cytotoxicity was analyzed by a trypan blue exclusion test. Experiments were performed in triplicate and data are shown as mean ± SD. Both cell lines showed dose-dependent effects with AE-SN treatment at 24 and 48 hr in the MTT assay and trypan blue exclusion test (one-way ANOVA, P < 0.001). AE-SN: aqueous extract of Solanum nigrum: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 2

AE-SN induced morphological changes and LC-3 activation in DLD-1 and HT-29 cells. (a) and (b), DLD-1 cells were treated with 0 or 1 mg/mL AE-SN for 48 hr, respectively; (c) and (d), HT-29 cells were treated with 0 or 1 mg/mL AE-SN for 48 hr. Arrows indicate lipid droplet-like morphological changes in AE-SN-treated cells. Magnification = 100x. (e) Total protein extracts were harvested from DLD-1 cells or HT-29 cells which were treated with 0 or 1 mg/mL AE-SN (control versus AE-SN) for 48 hr. Activation of caspase-3 and LC3 A/B in AE-SN-treated cells was determined by western blotting analysis. (f) Semiquantification of LC-3 A/B II in AE-SN-treated cells. Data presented are mean ± SD (n = 5). indicates statistical significance compared with 0 mg/mL AE-SN treatment using Student's t-test (P < 0.05). AE-SN: aqueous extract of Solanum nigrum; Con: control; GADPH: glyceraldehyde 3-phosphate dehydrogenase; LC-3 A/B II: mammalian microtubule-associated protein 1 light chain 3 A/B II.

Figure 3

Inhibition of AE-SN induced autophagy by using 3-MA, bafilomycin (a), and pepstatin A/E64d on DLD-1 and HT-29 cells. (a) DLD-1 and HT-29 cells were treated with 0.5 or 1.0 mg/mL AE-SN, respectively, in combination with 1 μM 3-MA, 2 nM bafilomycin A, or 2 μg/mL pepstatin A/E64d for 48 hr. Cytotoxicity was analyzed by MTT assay. Experiments were performed in triplicate and the data shown are mean ± SD. *indicates statistical significance compared with 0 mg/mL AE-SN treatment using Student's t-test (P < 0.05). (b) DLD-1 and HT-29 cells were treated with control, AE-SN (1 mg/mL), 3-MA, or AE-SN plus 3-MA (μM) for 48 hr and accumtrluation of LC3 A/B II was determined by Western blotting. Numbers indicated fold induction of LC3 A/B II compared with control. AE-SN: aqueous extract of Solanum nigrum; MTT: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.

Figure 4

Cytotoxicity of a combination of AE-SN and the chemotherapeutic drugs, cisplatin, doxorubicin, docetaxel, or 5-Fu. (a), (b), (c), and (d), DLD-1 cells were treated with 0 to 100 μM cisplatin, 0 to 10μM doxorubicin, 0 to 10 nM docetaxel, 0 to 100 μM 5-Fu in combination with 0, 0.2 or 5 mg/mL AE-SN for 48 hr; (e), (f), (g), and (h) HT-29 cells were treated with 0 to 100 μM cisplatin, 0 to 10μM doxorubicin, and 0 to 10 nM docetaxel, or 0 to 100 μM 5-Fu in combination with 0, 0.5 or 1 mg/mL AE-SN for 48 hr. Cytotoxicity was analyzed by MTT assay. Experiments were performed in triplicate and the data shown are mean ± SD. AE-SN: aqueous extract of Solanum nigrum; MTT: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.

Figure 5

Activation of cell death markers, LC3 A/B and caspase-3 in DLD-1 and HT-29 cells treated with AE-SN and cisplatin, doxorubicin, or docetaxel. Both DLD-1 and HT-29 cells were treated with 0, 1 mg/mL AE-SN, chemotherapeutic drugs alone, and a combination of 1 mg/mL AE-SN with chemotherapeutic drugs. The doses of cisplatin used were 50 μM in DLD-1 and 20 μM in HT-29. The dose of doxorubicin used was 5μM in DLD-1 and HT-29. The dose of docetaxel used was 1 nM in DLD-1 and HT-29, whereas the dose of 5-Fu was 20μM. After 48 hr incubation, total protein extracts were harvested from cells. (a) Activation of LC3 A/B and caspase-3 in DLD-1 and HT-29 cells was determined by western blotting analysis; (b) semiquantification of LC3 A/B II is presented as the relative fold induction of the control as mean ± SD (n = 3). *indicates statistical significance compared with 0 mg/mL AE-SN treatment using Student's t-test (P < 0.05). AE-SN: aqueous extract of Solanum nigrum; GADPH: glyceraldehyde 3-phosphate dehydrogenase; LC-3 A/B II: mammalian microtubule-associated protein 1 light chain 3 A/B II.

Figure 6

Proposed mechanism for the programmed cell death activated by AE-SN and chemotherapeutic drugs in human colorectal carcinoma cells. AE-SN-activated cell death can increase with cisplatin/doxorubicin/docetaxel/5-Fu-activated caspase-3-dependent or independent cell death (+/− caspase-3 cleavage) and result in further enhanced tumor cell death. (+) indicates synergistic effect of AE-SN with chemotherapeutic drugs. Solid line: activated pathway; the dotted line: inactive pathway. AE-SN: aqueous extract of Solanum nigrum.