Abstract
Colorectal cancer was the third most commonly diagnosed malignant tumor and the fourth leading cause of cancer deaths worldwide in 2012. A human colorectal cancer cell line, RCM-1, was established from a colon cancer tissue diagnosed as a well-differentiated rectum adenocarcinoma. RCM-1 cells spontaneously form 'domes' (formerly designated 'ducts') resembling villiform structures. Two sulphur-containing compounds from Cucumis melo var. conomon (Katsura-uri, or Japanese pickling melon), referred to as 3-methylthiopropionic acid ethyl ester (MTPE) and methylthioacetic acid ethyl ester (MTAE), can induce the differentiation of the unorganized cell mass of an RCM-1 human colorectal cancer cell culture into a dome. However, the underlying molecular mechanisms of such dome formation have not been previously reported. Here, we performed a structure-activity relationship analysis, which indicated that methylthioacetic acid (MTA) was the lowest molecular weight compound with the most potent dome-inducing activity among 37 MTPE and MTAE analogues, and the methylthio group was essential for this activity. According to our microarray analysis, MTA resulted in down-regulation of 537 genes and up-regulation of 117 genes. Furthermore, MTA caused down-regulation of many genes involved in cell-cycle control, with the cyclin E2 (CCNE2) and cell division cycle 25A (CDC25A) genes being the most significantly reduced. Pharmacological analysis showed that the administration of two cell-cycle inhibitors for inactivating CDC25A phosphatase (NSC95397) and the cyclin E2/cyclin-dependent kinase 2 complex (purvalanol A) increased the dome number independently of MTA. Altogether, our results indicate that MTA is the minimum unit required to induce dome formation, with the down-regulation of CDC25A and possibly CCNE2 being important steps in this process.