Cordycepin inhibits colon cancer proliferation by suppressing MYC expression

Cordycepin is a purine nucleoside anti-metabolite and anti-biotic isolated from the fungus Cordyceps militaris, which has potential anti-neoplastic activities. This study aimed to investigate the effect of cordycepin in inhibiting colon cancer development. The proliferation of cordycepin-treated HCT116 and Caco-2 colon cancer cell lines was assessed with 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the viability was measured with colony formation assay. At the same time, cordycepin responsive gene and microRNAs (miRNAs, miRs) were screened by qRT-PCR. MYC over-expressing HCT116 and Caco-2 cell lines were constructed, which were further transfected with miR-26a. Inhibitory effect of cordycepin on cell proliferation was evaluated with cell viability assay, cell number count, and colony formation assay. The relative expression of MYC and miR-26a was detected by qRT-PCR and Western blot. Cordycepin inhibited colon cancer cell proliferation by down-regulating MYC mRNA/protein expression and up-regulating miR-26a in both HCT116 and Caco-2 cells. MYC over-expression could suppress the expression of miR-26a, which could be restored by cordycepin treatment. Additional miR-26a transfection in MYC over-expressing cells could reverse MYC over-expression-promoted proliferation, which could be further potentiated by cordycepin treatment. Cordycepin is able to suppress colon cancer cell proliferation, likely mediated by the MYC/miR-26a pathway, supporting its potential for the treatment of colon cancer.


Introduction
As the third most common digestive tract cancer, colon cancer ranks the second in mortality globally. Despite significant improvements in conventional therapy, the five-year survival rate remains below 20% due to frequent recurrence and metastasis [1,2]. The high inter-patient variability, manifested by genomic heterogeneity, makes targeted therapies less reliable [3,4]. While it is worth noting that, as a proto-oncogene and a classical Wnt pathway target gene, enhanced and/or altered expression of MYC expression are universally present in colon cancer [5]. MYC deletion could suppress tumorigenesis in both syngeneic and humanized mouse models [6,7]. Given the lack of promising chemotherapeutic drugs for MYC, significant research attention has been invested to inhibit the expression or activity of MYC [8].

Colony formation assay
Transfected or un-transfected Caco-2 and HCT-116 cells were cultured in 6-well plates (1 × 10 3 cells per well) for two weeks, which were further fixed with 4% paraformaldehyde and stained with crystal violet. The number of colonies was counted to assay the in vitro cell survival.

qRT-PCR analysis
Total RNAs were extracted from colon cancer cells using the TRIzol reagent (Invitrogen) and reverse transcribed into cDNA with the PrimeScript RT reagent Kit (Takara, Dalian, China) and One Step PrimeScript miRNA cDNA Synthesis Kit (Takara). SYBR Green Real-time PCR Master Mix (Takara) was utilized to detect the amplification (95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min) on an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA). The relative expression was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6 and calculated using the 2 -ΔΔCt method. The primers for the mRNA detected were listed as follows: . Primers for microR-NAs (miRNAs, miRs) were ordered from Merck (Kenilworth, NJ), including miR-26a-5p, miR-26b, miR-92a, miR-29b, miR-34a, and U6.

Western blot
The cell lysate was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nylon membranes, which was incubated with MYC primary antibody (Abcam, 1:1000 dilution) at 4 °C overnight, and further incubated with a peroxidaseconjugated secondary antibody (Sigma-Aldrich, 1:1000 dilution) at room temperature for 2 h and developed with an ECL system (GE. Healthcare Life Sciences). The relative expression of MYC was normalized with β-actin (Santa Cruz, Dallas, TX) using NIH-Image J1.51.

Statistical analysis
Student's t-test and one-way or two-way ANOVA analysis were used for statistical analysis, and the significance level was set as p-value < 0.05. All statistical analyses were performed with GraphPad Prism (GraphPad Software, Inc., San Diego, CA).

Cordycepin inhibits the proliferation of colon cancer
Cordycepin exhibited a dose-dependent and timedependent inhibitory effect on viability of HCT116 cells (Fig. 1A) and Caco-2 cells (Fig. 1B) as measured by the MTT assay. The half-maximal inhibitory concentration (IC 50 ) was less than 100 μM after 72 h incubation, therefore 100 μM cordycepin was chosen in the following experiments. The decreased total cell number counted ( Fig. 2A) and the number of colony formation (Fig. 2B, P < 0.01) further verified the inhibitory effect of cordycepin on the colon cancer cell lines.

Cordycepin suppresses MYC expression to inhibit the proliferation of colon cancer
Cordycepin response genes were screened by qRT-PCR, and among the screened genes of interested (MYC, MYB, JUN, FOS, STAT3, TFAP2A, E2F1, and GATA3), MYC was the only one down-regulated upon cordycepin treatment (Fig. 3A). The protein expression of MYC was also down-regulated after cordycepin treatment as indicated by Western blot analysis (Fig. 3B). In order to study the role of MYC in colon cancer cells, MYC over-expressing HCT116 and Caco-2 cells (Fig. 3C) were constructed. MYC over-expression could significantly promote the proliferation of both HCT116 and Caco-2 cells as indicated by the cell number count (Fig. 3D), cell viability assay (Fig. 3E), and colony formation assay (Fig. 3F), all of which were significantly inhibited by cordycepin treatment. These results indicated that MYC might be the cordycepin response gene to mediate its inhibitory effect in colon cancer.

Discussion
Polyadenylation is a vital process to produce mature mRNA for translation, which can activate AMP-activated protein kinase (AMPK) and suppress the mammalian target of rapamycin (mTOR) signaling pathway [16]. As a polyadenylation inhibitor, cordycepin promotes apoptosis and inhibits proliferation of tumor cells. The dissociation of MYC mRNA/protein expression is reported in HeLa 1C5 cells and human diploid fibroblastic cell line FS-4 [15], where MYC proteins do not follow the reduced expression of its mRNA after cordycepin administration. While such dissociation is not observed in HCT116 cells and Caco-2 cells, whether this dissociation is a universal mechanism needs to be further investigated. MYC dysregulation is associated with aggressive biological behavior and adverse clinical outcome of colon cancer [17]. Increasing evidence has indicated that MYC induces widespread miRNA repression, while its own activity could also be regulated by miRNAs [18]. In Burkitt lymphoma, MYC can stimulate enhancer of zeste homolog 2 (EZH2) expression by suppressing its negative regulator miR-26a [19]. While in glioblastoma multiforme, MYC could directly increase miR-26a expression to regulate the tumor suppressor phosphatase and tensin homolog (PTEN) [20]. It is worth noting that miR-26a could suppress MYC by targeting the Wnt pathway coactivator, cyclin-dependent kinase 8 (CDK8), to inhibit progression and metastasis of hepatocellular carcinoma [21]. Whether miR-26a could mediate MYC inhibition to complete the full MYC/miR-26a regulatory loop in colon cancer needs to be further studied.
Some study limitations should be indicated. It is generally accepted that, as a transcriptional factor without a suitable pocket for high-affinity binding, MYC is undruggable by low molecular weight inhibitors [22]. Cordycepin can be utilized to down-regulate the expression of MYC, while the precise interaction mechanism is still not understood. MiR-26a can promote the proliferation and tumorigenesis of ovarian cancer, as well as the invasion and metastasis of hepatocellular carcinoma [23,24]. Consistently, we also observed in our study that cordycepin could inhibit the proliferation of colon cancer. However, potential effect of cordycepin on apoptosis, as well as therapy resistance, should be investigated by future study.
In this study, we found that MYC mRNA/protein expression could be inhibited by cordycepin, while miR-26a could be up-regulated by cordycepin. MYC could repress the function of miR-26a to mediate the effect of cordycepin. Our study proposes the clinical potential of cordycepin in treating colon cancer by targeting the MYC/miR-26a pathway. MYC/miR-26a pathway is critical for cordycepin-mediated proliferation suppression. A HCT116 cells and B Caco-2 cells were incubated with or without cordycepin, and the cell proliferation was determined using cell number assay. C HCT116 cells and (D) Caco-2 cells were incubated with or without cordycepin for 72 h, and the cell proliferation was determined using MTT assay. E HCT116 cells and (F) Caco-2 cells were subjected to colony formation assay with or without cordycepin for two weeks. Cordycepin + and cordycepin free represent the culture media with and without 100 μM/ml cordycepin, respectively. Data were mean ± S.D. of three independent experiments, and each measured in triplicate (**p < 0.01)

Conclusion
Cordycepin could be considered as a treatment option for colon cancer by regulating the MYC/miR-26a pathway.