Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model
Introduction
Colorectal cancer (CRC) is the second most prevalent cancer and the third leading cause of cancer deaths worldwide. Surgery is the primary treatment for CRC. However, most patients with metastasis are candidates for systemic chemotherapy to palliate symptoms and prolong life.1, 2 At the present time, 5-fluorouracil (5-FU) remains the cornerstone of systemic treatment for colorectal cancer. 5-FU has been widely used for stage Ш colon cancer and stage Ш rectal cancer since the 1980s. In the 1990s, the 5-FU/leucovorin protocol was accepted as a standard for CRC treatment.3, 4 However, along with its usage, resistance to 5-FU has become common and has been recognised as a reason for CRC therapy failure. Although many aggressive adjuvant therapies such as irinotecan and oxaliplatin combined with 5-FU are being tested in clinical treatment, a novel chemotherapy combination still needs to be explored.5
Autophagy is an evolutionary conserved eukaryotic process in which organelles and bulk proteins are turned over by lysosomal activity. The most distinctive feature of autophagy is the formation of autophagosomes, double- membrane vesicles that fuse with lysosomes for hydrolytic cleavage of engulfed proteins and organelles.6 In mammalian cells, microtubule-associated protein 1 light chain 3 (LC3) is conjugated to phosphatidyl ethanolamine for insertion into the autophagosome membrane. LC3-II (a lipidated form of LC3) has been deemed a marker of autophagy and detecting LC3-II by immunoblotting or immunofluorescence is a reliable method for monitoring autophagosome formation.7, 8 Moreover, p62 protein has recently been reported as an autophagy substrate whose level decreases upon autophagy induction; as such, it has been used to monitor autophagy flux.8, 9
The physiologic function of autophagy is to maintain homeostasis by eliminating unnecessary proteins and injured or aged organelles. Recently, increasing evidence has shown that autophagy is also associated with many pathologic conditions such as neurodegenerative diseases, hereditary myopathies, and cancer.10 Many studies have focused on the relationship between autophagy and tumour pathogenesis, development, and treatment. But autophagy seems to play a paradoxical role in cancer cell survival and death.11, 12, 13 Moreover, chemotherapy also induces autophagy in cancer. Because studies about autophagy improving survival or inducing death both exist,14, 15, 16, 17 whether autophagy enables cells to survive or enhances their death is context-driven and depend on the stimuli type, nutrient availability, organism development, and apoptotic status.18, 19
In our previous study, we found that the autophagy inhibitor 3-MA enhanced the effect of 5-FU-induced apoptosis in 2 colon cancer cell lines, HT29 and colon26.20 This suggested that targeting autophagy inhibition could be a promising strategy during 5-FU chemotherapy in colon cancer. However, since colon cancer is heterogeneous,21 the application is limited. So, in this study, three other kinds of human colon cancer cells were used. To more directly detect the role of autophagy, Atg7 small interfering RNA (siRNA) were also applied and the resulting change of 5-FU effects was observed. Furthermore, DLD-1 xenografts in nude mice were used to confirm the role of autophagy in vivo.
Section snippets
Cell culture
The human colorectal carcinoma HCT116 and DLD-1 cell lines were obtained from American Type Culture Collection. The 5-FU-resistant sub-line of the DLD-1 cell line (DLD-1/5-FU) was established by repeated, continuous exposure to 5-FU with stepwise escalation as described previously.22 All cell lines were cultured with RPMI-1640 (Wako, Japan) supplemented with 10% foetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37 °C in a humidified atmosphere of 95% air and
5-FU-induced colon cancer cell death was enhanced by 3-MA treatment
Three kinds of human colon cancer cells, HCT116, DLD-1, and DLD-1/5-FU (a 5-FU–resistant sub-line of DLD-1), were used and the IC50 for 48-h 5-FU treatment was examined via MTT assay. The IC50 in HCT116, DLD-1, and DLD-1/5-FU was 7.09 ± 0.87 μM, 47.25 ± 5.97 μM, and 2149 ± 160 μM, respectively (Supplementary Fig. S1). As a result, we used 7 μM of 5-FU in HCT116 and 50 μM in DLD-1 and DLD-1/5-FU for 48 h in the subsequent experiment.
Next, changes in colon cancer cell death were examined using 5-FU with or
Discussion
In colorectal cancer, chemotherapy is currently used to reduce tumour recurrence and prolong patients’ lives. However, due to drug resistance, exploration of new chemotherapy strategies is very important. From our previous report,20 we found that the autophagy inhibitor 3-MA could enhance 5-FU–induced HT29 (a human colon cancer cell line) and colon26 (a mouse colon cancer cell line) cell death (including apoptosis). In this study, we used 2 other human colon cancer-derived cell lines (HCT116
Conflict of interest statement
None declared.
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