Study On The Molecular Mechanism By Which Tolfenamic Acid Suppressed The Growth Of Colorectal Cancer Cells

Colorectal cancer is one of the most common malignancy. According to GLOBOCAN2008data, the incidence and mortality rate of colorectal cancer worldwidely rank top three inboth men and women. Given the improvement of material living conditions and aggravationof environmental pollution, the incidence of colorectal cancer could be increased for the nextyears in our country. In fact, colorectal cancer is curable at the early stages of development,and is essentially incurable in its advanced stage. Epidemiological and clinical studies suggestthat non-steroidal anti-inflammatory drugs (NSAIDs) are potent chemopreventive agents forcolorectal cancer. Tolfenamic acid (TA) is one of NSAIDs which is widely used in humansand other species. Compared with other similar drugs, TA harbors better anti-inflammatoryeffect and lower toxicity. It was paid more attention due to recent findings that TA suppressedtumorigenesis in different cancer models, including colorectal cancer. Cyclin D1, as a keyregulator of cell cycle factors, is often aberrantly expressed in most cancer cells, associatedthe occurrence and development of tumor. In current study, the effect of tolfenamic acid onthe expression of cyclin D1protein was first studied. Then we mainly investigated thesignaling pathways involved in that tolfenamic acid regulated cyclin D1expression, and therelationship between signling pathway activation and apoptosis in colon cancer cells. Thefollowing results were obtained here:1. The effect of TA on the cyclin D1expression was analyzed in cancer cells. As as aresult, cyclin D1was overexpressed in colorectal cancer cells and tissues. TA can effectivelyreduce cyclin D1expression in various cancer cells, including colorectal cancer cells, as wellas in colorectal tumors. Under the condition of same dose, TA worked better in term ofinhibition of cyclin D1expression compared with other commonly used NSAIDs, withexception of sulindac sulfide which is usually used for the treatment of colorectal polyps.Furthmore, TA significantly suppressed the phosphorylation of retinoblastoma protein intime-dependent manner in colon cancer cells (P<0.05), which could be, at least in part,mediated by downregulation of cyclin D1. In addition, post24h treatment with TA (50μM)resulted in a significant increase in G2-M phase of HCT116cell cycle (P<0.01), which could contribute to inhibition of cell proliferation.2. To investigate the regulation mechanism of cyclin D1down-regulation by TA, cyclinD1gene transcription and protein stability was evaluated. The results showed that thetranscription levels of cyclin D1gene expression was not affected by1h treatment with TA,whereas there was reduction in cyclin D1mRNA expression in the presence of TA for24h.Moreover, the expression of transcription factors (such as β-catenin, Sp-1, etc.) wassuppressed by24h treatment with TA, which might be responsive for decrease in cyclin D1promoter activity (P<0.01), thus affecting cyclin D1transcription. On the other hand, after theaddition of cycloheximide (CHX,10μg/mL), the rate of decrease in cyclin D1proteinexpression did not significantly change between control group and TA treatment group,indicating protein stability was not affected by TA. In addition, different inhibitors of proteindegradation pathways did not restore cyclin D1expression in the presence of TA. Bothphosphorylation (Thr286) and subsequent unbiquitination of cyclin D1protein was notincreased by TA. Collectively, these data have shown that TA rapidly down-regulats cyclin D1expression which occurs at neither the transcriptional nor post-translational level, thereforesuggesting that TA inhibits cyclin D1protein bio-synthesis.3. The effect of TA on mTOR-mediated signaling pathways was investigated. As resultsshown, TA treatment activated upstream positive regulatory factor—Akt protein kinase wasactivated, and inactivated negative regulatory factor—GSK-3β protein kinase of mTORsignaling pathway. Meantime, its downstream effector p70S6kinase (threonine389) and S6protein (serine235/236) were significantly phosphorylated in time-dependent manner(P<0.01), indicating the activation of mTOR-mediated signaling pathway by TA. Given theactive mTOR signal is contradictory with the inhibition of cyclin D1protein translation by TA,there could be some other pathway(s) involved in this process.4. We analyzed the effect of TA on endoplasmic reticulum (ER) stress responses and itsrelationship with cyclin D1down-regulation. We observed that TA could significantlyincrease the transcriptional activity of ATF6transcription factors (P<0.01), and induce XBP-1gene mRNA splicing, suggesting that XBP-1protein transcriptional activity increased. Inaddition, a variety of marker gene expression of ER stress responses was induced by TA, suchas BiP, ATF4, CHOP, etc. All these data were consistently obtained from two different coloncancer cell lines—HCT116and SW480cells, and showed TA activated ER stress-mediatedunfolded protein response (UPR) pathways. Correspondingly, TA led to increasedphosphorylation of eIF2α proteins in HCT116cells, accompanied with down-regulation ofcyclin D1protein. Silence of PERK which mediated eIF2α phosphorylation when cellssuffered from ER stress significantly restored cyclin D1expression in the presence of TA (P<0.01), showing PERK/eIF2α signaling pathway was involved in inhibition of cyclin D1protein.5. Whether UPR signal activation is associated with TA-mediated apoptosis was studied.The data showed that Silencing PERK expression resulted in a reduction in TA-mediatedeIF2α phosphorylation, subsequently proapoptotic protein—ATF4and CHOP expression,indicating the activation of PERK/eIF2α/ATF4/CHOP axis which is a branch of UPRsignaling pathway. Moreover, TA suppressed anti-apoptotic protein Bcl-2expression,promoted PARP cleavage, thereby inducing apoptosis. However, silence of ATF4attenuatedTA-mediated apoptosis evaluated by cleaved PARP expression. The data indicated that theactivation of these signaling pathways played a role in tolfenamic acid-induced apoptosis incancer cells.Taken together, the present study found that TA activated ER stress responses, at least incolon cancer cells, which resulted in various signaling pathways’ activation, thus differentiallyregulating the expression of anti-apoptotic protines such as cyclin D1and Bcl-2, as well aspro-apoptotic proteins such as ATF4and CHOP, which, at least in part, finially contributed tocell growth inhibiton by TA. We thoroughtly investigated the regulation mechanism of cyclinD1expression by TA in colon cancer cells, and showed evidence that TA activated UPRsignaling pathways, providing a new theoretical basis for further understading its anti-canceraction.