Lowering the apoptotic threshold in colorectal cancer cells by targeting mitochondria

Abstract

Background: Colorectal cancer is the third most-common cancer and the second most-common cause of cancer related death in UK. Although chemotherapy plays significant role in the treatment of colorectal cancer, morbidity and mortality due to drug resistance and cancer metastasis are yet to be eliminated. Recently, doxycycline has been reported to have cytotoxic and anti-proliferating properties in various cancer cells. In this study, whether doxycycline was apoptosis threshold lowering agent in colorectal cancer cells by targeting mitochondria was answered.

Results: This study showed dose-dependent cytotoxic effects of cisplatin, oxaliplatin and doxycycline in HT29 colorectal cancer cells. Doxycycline showed inhibition of cytochrome-c-oxidase activity in these cells over a time-period. The pre-treatment of doxycycline reported statistically significant increased cytotoxicity of cisplatin and oxaliplatin compared to cisplatin and oxaliplatin alone. The caspase studies revealed significantly less expression and activity of caspase 3 in HT29 cells pre-treated with doxycycline compared to the cells treated with cisplatin and oxaliplatin alone.

Conclusions: It was concluded that doxycycline lowered the apoptotic threshold in HT 29 cells by targeting mitochondria. This also raised possible caspase-independent mechanisms of apoptosis in HT29 cells when pre-treated with doxycycline however this needs further research work.

Figures

Figure 1

Dose response relationship of cisplatin, oxaliplatin and doxycycline. 2 × 104 HT 29 cells/ml were treated with various concentrations (1 to 1000 micromolar) of cisplatin, oxaliplatin and doxycycline (0.1 to 100 micrograms/ml) for 24 hours in the wells of 24 well plates and the cytotoxicity measured with Alamar blue assay according to the manufacturer's instructions. Control was taken as untreated cells. Data are depicted as means of six experiments ± standard deviation.

Figure 2

Dose response relationship of combination treatment of doxycycline with platinum compounds. 2 × 104 HT 29 cells/ml were treated in the wells of 24 well plates with cisplatin or oxaliplatin with and without 10 μg/ml of doxycycline for 24 hours. The cytotoxicity was assessed with Alamar blue assay according to the manufacturer's instructions. Data are depicted as means of six experiments ± standard deviation. (p = 0.84 for cisplatin and p = 0.95 for oxaliplatin experiments).

Figure 3

Cytochrome c oxidase activity. 2 × 104 HT 29 cells were treated with 10 μg/ml of doxycycline over a period of 4 days and staining for cytochrome c oxidase activity was performed as mentioned in methods. The brown staining is indicative of cytochrome c oxidase activity, while purple staining is indicative of cell nucleus. Representative micrographs of six independent experiments are shown. Scale bar equals 20 μM.

Figure 4

Comparative cytotoxicity and anti-proliferative effects of doxycycline. 2 × 104 HT 29 cells/ml were treated with doxycycline for 24 hours and 3 days in the wells of 6 well plates. The cytotoxicity and cell proliferation studies were performed with Alamar blue assay and Picogreen assay according to the manufacturer's instructions respectively. Data are depicted as means of six experiments ± standard deviation. (* - Denotes statistically significant difference in the activity).

Figure 5

Pre-treatment of doxycycline. 2 × 104 HT 29 cells/ml were treated with 10 μg/ml of doxycycline for 3 days in the wells of 6 well plates followed by treatment with different concentrations of cisplatin and oxaliplatin for 24 hours. In similar settings, another set of experiments was established without pre-treatment of doxycycline for 3 days. The cytotoxicity was assessed with Alamar blue assay according to the manufacturer's instructions. Data are depicted as means of six experiments ± standard deviation. (p = 0.003 for cisplatin and p = 0.003 for oxaliplatin experiments). (* - Denotes statistically significant difference in the activity).

Figure 6

Caspase 3 gene expression. Gel electrophoresis of GAPDH amplicons is displayed as control on the left side of figure. (CON = Control, DOC = Doxycycline, CIS = Cisplatin, CD = Cisplatin and Doxycycline, OXA = Oxaliplatin, OD = Oxaliplatin and Doxycycline). The right side of figure displays gel electrophoresis of caspase 3. (NC = Negative control, Con = CON = Positive control, Dox = DOX = Doxycycline, C(CIS)1; C4; C24 = Cisplatin treatment for 1, 4 and 24 hours respectively, cd(CD)1; cd4; cd24 = Cisplatin and Doxycycline treatment for 1, 4 and 24 hours respectively, O(OXA)1; O4; O24 = Oxaliplatin treatment for 1, 4 and 24 hours respectively, od(OD)1; od4; 0d24 = Oxaliplatin and Doxycycline treatment for 1, 4 and 24 hours respectively). Data are depicted as means of six experiments ± standard deviation. (* - Denotes statistically significant difference in the activity).

Figure 7

Real time PCR for caspase 3 gene expression. This figure displays quantitative presentation of caspase 3 gene expression in HT 29 cells by real time PCR. (CON = control, DOX = doxycycline, CIS1; 4; 24 = Cisplatin treatment for 1, 4 and 24 hours respectively, CD1; 4; 24 = Cisplatin and Doxycycline treatment for 1, 4 and 24 hours respectively, OXA1; 4; 24 = Oxaliplatin treatment for 1, 4 and 24 hours respectively, OD1; 4; 24 = Oxaliplatin and Doxycycline treatment for 1, 4 and 24 hours respectively). Data are depicted as means of six experiments ± standard deviation. (* - Denotes statistically significant difference in the activity).

Figure 8

Caspase 3 activity. This figure displays quantitative assessment of caspase 3 activity in HT 29 cells following drug treatments. (CON = control, DOX = doxycycline, C1; 4; 24 = Cisplatin treatment for 1, 4 and 24 hours respectively, CD1; 4; 24 = Cisplatin and Doxycycline treatment for 1, 4 and 24 hours respectively, O1; 4; 24 = Oxaliplatin treatment for 1, 4 and 24 hours respectively, OD1; 4; 24 = Oxaliplatin and Doxycycline treatment for 1, 4 and 24 hours respectively). Data are depicted as means of six experiments ± standard deviation. (* - Denotes statistically significant difference in the activity).