Colorectal cancer constitutes the third most common cancer in UK. It is the second leading cause of cancer deaths in UK. About one in 20 develops colorectal cancer in their lifetime. About 16,000 people are dying from the colorectal cancer every year in UK (Cancer Statistics, Cancer Research UK 2005). Three main therapeutic options available for colorectal cancer; Surgery, Chemotherapy and Radiotherapy, are used either on their own or in combinations. The main reasons for mortality from colorectal cancer are metastases or recurrence. Recently, doxycycline has been shown to have MMPs inhibitory actions and thus has potential in prevention of metastases[
14]. Apart from being an antibiotic, doxycycline has been reported to have cytotoxic actions and to suppress cell proliferation[
14]. To date, various studies have reported the apoptotic effects of doxycycline in different cancers including prostate cancer[
16], leukaemia[
6], osteosarcoma[
17] and pancreas cancer[
18]. However, precise mechanisms of apoptosis are not well understood. Similarly, none of the work has investigated the impact of doxycycline with other platinum agents in colorectal cancer. This study answered whether doxycycline has any synergistic effects with platinum compounds; cisplatin and oxaliplatin, exploring its potential use in colorectal cancer therapy.
Results of this study showed cisplatin, oxaliplatin and doxycycline to be cytotoxic to HT 29 colorectal cancer cells following 24 hours of treatment in a dose-dependent manner; however, cisplatin and oxaliplatin were more cytotoxic than doxycycline (figure
1). To rule out any impact of serum in media, the cytotoxicity studies were performed using media with and without serum. It was found that the drugs were more cytotoxic in media containing serum, but this did not reach statistical significance (data not shown) and therefore future experiments were performed in the serum supplemented media. The combination treatment of doxycycline and platinum compounds in HT29 cells did not show any statistically significant difference in the cytotoxicity compared to cisplatin and oxaliplatin alone following 24 hours of treatments (figure
2). Thus in these initial experiments, our study failed to show any synergistic actions of doxycycline with platinum compounds. As these combinations did not show any difference in the cytotoxicity, the duration of treatment with doxycycline necessary to decrease the threshold for the cytotoxic effects of the cisplatin and oxaliplatin were looked in. As cytochrome
c oxidase (complex IV) plays significant role in the oxidative phosphorylation of mitochondria and doxycycline works by inhibition of mitochondrial protein synthesis and thus inhibition of oxidative phosphorylation, cytochrome
c oxidase activity was assessed over a period of time following doxycycline treatment. The cytochrome c oxidase activity reduced significantly from day 3 onwards in HT 29 cells following treatment of doxycycline (Figure
3). Following this finding, three days treatment of doxycycline of HT 29 cells showed significantly increased cytotoxic and anti-proliferative effects compared to 24 hours of treatment (figure
4). Thus it was decided to pre-treat HT 29 cells for 3 days before the treatment of platinum compounds to assess whether this would help to lower the apoptotic threshold. The cytotoxicity studies following 3 day pre-treatment of doxycycline revealed statistically significant higher cytotoxicity compared to cisplatin or oxaliplatin treatment alone (Figure
5). This suggested that doxycycline lowered the apoptotic threshold in colorectal cancer cells for the cytotoxic actions of cisplatin and oxaliplatin. Although doxycycline had shown apoptotic lowering potential, the precise mechanism of this effect was unclear. In an attempt to understand it, the caspase studies were performed. Caspases are the cysteine proteases and are crucial for the process of apoptosis. Caspase 3 is one of the executioner caspases involved towards the end of apoptotic induction following intrinsic as well as extrinsic pathways of apotptosis. Caspase 3 gene expression study revealed increased gene expression in a dose dependent manner in HT 29 cells treated with cisplatin and oxaliplatin but there was no increase in caspase 3 gene expression in the cells pre-treated with doxycycline (figure
6). These findings were confirmed by another method for assessment of caspase 3 gene expression (real time PCR studies) (figure
7). In order to verify these findings, it was decided to look at caspase 3 activity in these cells. The caspase 3 activity study reported similar findings to the caspase 3 gene expression study (figure
8). The reduction in the caspase 3 gene expression and activity in HT 29 cells pre-treated with doxycycline might be due to the time change in the measurement of gene expression and activity. However, caspase-independent mechanisms of apoptosis might be another explanation for these findings as few studies reported caspase-independent mechanisms of apoptosis in different cell lines[
15]. Thus, this study suggested that pre-treatment of doxycycline lowered the apoptotic threshold in HT 29 colorectal cancer cells which might be due to caspase-independent mechanisms.
The advantage of doxycycline over other tetracyclines is its longer duration of action and comparatively less toxicity. Apart from cytotoxic and anti-proliferative properties of doxycycline, the key finding in this experiment was the inhibition of cytochrome c oxidase by doxycycline which suggested involvement of mitochondria in its actions. This study also suggests beneficial role of doxycycline pre-treatment in enhancing the cytotoxic effects of cisplatin and oxaliplatin with possible caspase independent mechanisms. As deficits or defects in the apoptotic pathways are frequently present in the tumours and are possible reasons for resistance to the chemotherapeutic agents, doxycycline may be useful in overcoming drug resistance however further research is required to evaluate the precise role of doxycycline in more detail before considering its potential use in clinical trails.