Abstract
Elucidation of the molecular mechanisms by which 5-fluorouracil (5-FU) induces apoptosis is required in order to understand the resistance of colorectal cancer (CRC) cells to 5-FU. In the current study, 5-FU-induced apoptosis was assessed using the propidium iodide method. Involvement of protein kinase C (PKC) was assessed by evaluating the extent of their activation in CRC, following treatment with 5-FU, using biochemical inhibitors and western blot analysis. The results revealed that 5-FU induces varying degrees of apoptosis in CRC cells; HCT116 cells were identified to be the most sensitive cells and SW480 were the least sensitive. In addition, 5-FU-induced apoptosis was caspase-dependent as it appeared to be initiated by caspase-9. Furthermore, PKCɛ was marginally expressed in CRC cells and no changes were observed in the levels of cleavage or phosphorylation following treatment with 5-FU. The treatment of HCT116 cells with 5-FU increased the expression, phosphorylation and cleavage of PKCδ. The inhibition of PKCδ was found to significantly inhibit 5-FU-induced apoptosis. These results indicated that 5-FU induces apoptosis in CRC by the activation of PKCδ and caspase-9. In addition, the levels of PKCδ activation may determine the sensitivity of CRC to 5-FU.
Highlights
Colorectal cancer (CRC) is the third most common malignancy in the populations of developed western countries and represents the third leading cause of cancer‐associated mortality in the USA [1]
Whole cell lysates were subjected to western blot analysis and western blot analysis of the GAPDH levels was included to demonstrate that equivalent amounts of protein were loaded in each lane
Data are representative of two individual experiments. 5‐FU, 5‐fluorouracil; PKC, protein kinase C; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase
Summary
Colorectal cancer (CRC) is the third most common malignancy in the populations of developed western countries and represents the third leading cause of cancer‐associated mortality in the USA [1]. Once the disease spreads to distant sites, it is usually incurable using the current systemic treatment options, including chemotherapy This is largely due to the resistance of cancer cells to apoptosis. The chemotherapeutic agent, 5‐fluorouracil (5‐FU), induces cytotoxic effects via targeting the metabolism of RNA bases [3]. It is predominantly administered in the treatment of various types of cancer, including breast, aerodigestive tract and head cancer. The protein kinase C (PKC) family includes 11 distinct members, which share a similar serine (Ser)/threonine (Thr) structure [9,10] These polypeptide isoenzymes are classically classified into three distinct groups according to the second messengers to which they respond. PKCε is an antiapoptotic enzyme, which promotes cell proliferation and resistance to chemotherapeutic agents [12], whereas PKCδ is classified among the proapoptotic PKCs as its cleavage and activation promotes apoptotic cell death [13]
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