Inhibition of Colorectal Cancer Cell Proliferation by Regulating Platelet-Derived Growth Factor B Signaling with a DNA Aptamer

Background: Overexpression of platelet-derived growth factor-BB (PDGF-BB) is associated with colorectal carcinogenesis. PDGF-BB plays a role in the autocrine growth stimulation of cancer cells. Aptamers are short single-stranded oligonucleotides that can bind to cellular targets with high affinity and specificity and offer the advantage of non-immunogenicity, non-toxicity and high stability. Thus, they receive interest as potential therapeutic agents. Methods: The endogenous level of PDGF-BB in Caco-2 and SW480, colorectal cancer (CRC) cells, was evaluated using ELISA. The effect of the PDGF-BB aptamer on cell proliferation was investigated in two CRC cell lines and CCD841 CoN, normal colon cells. The effective molar ratio between PDGF-BB and PDGF-BB aptamer was further explored. Cell viability in all experiments was analyzed using MTS assay. Western blotting was performed to examine the alteration of relevant signaling pathways. Results: Caco-2 and SW480 cells endogenously synthesized and secreted PDGF-BB to stimulate their growth. Cells treated with the PDGF-BB aptamer proliferated at a slower rate, but CCD841 CoN did not. Pre-incubation of PDGF-BB with the corresponding aptamer at the molar ratio 1:1 could significantly silence its proliferative effect on CRC cells. Western blot analysis revealed that the phosphorylation level of ERK1/2, a key component in PDGF downstream signaling pathway, was down-regulated by the aptamer, indicating the underlying mechanism of inhibition of CRC cell proliferation. Conclusions: This study demonstrated that using a DNA aptamer to interfere with the binding of PDGF-BB to its receptor suppressed CRC cell proliferation in part via down-regulation of the Ras/Raf/MEK/ERK signaling pathway. It raised the possibility that the PDGF-BB-specific aptamer could be a promising therapeutic agent for CRC targeted therapy.

Comparison of aspirin response measured by urinary 11-dehydrothromboxane B2 and VerifyNow aspirin assay in patients with ischemic stroke.

BACKGROUND: We looked for the prevalence of aspirin nonresponders, compared the results of 2 tests assessing aspirin responses-measurement of urinary 11-dehydrothromboxane B2 (dTXB2) and VerifyNow Aspirin assay-in patients with ischemic stroke, and examined the relationship of aspirin nonresponse and the outcomes of the patients. METHODS: One hundred one patients with ischemic stroke were prospectively included. Aspirin response was assessed by urinary dTXB2 measurement and VerifyNow Aspirin assay. The Spearman correlation coefficients and kappa statistics were calculated to assess correlation and agreement between the 2 tests. The measured outcome was the occurrence of cardiovascular events and death. RESULTS: Prevalence of aspirin nonresponders was 40% and 6%, if they were measured by urinary dTXB2 and VerifyNow Aspirin assay, respectively. Poor correlation in the results between the 2 tests was found (r = .135, P = .190). The degree of agreement between the 2 tests in relation to resistance status was weak (kappa = .032, P = .590). With a mean follow-up time of 17 months, the outcomes occurred significantly higher in aspirin nonresponders who were diagnosed by urinary dTXB2 measurement as compared with patients with aspirin response (18% versus 2%, odds ratio 8.8, 95% confidence interval 1.18-65.4, P = .037). CONCLUSIONS: Our research confirmed poor correlation and lack of agreement between the 2 tests. Only aspirin nonresponders who were diagnosed by dTXB2 measurement were related to having cardiovascular events and death. Further research is still needed to identify the best method of diagnosis of aspirin nonresponders.

Aspirin nonresponders in patients with ischaemic stroke.

From previous studies, the prevalence of aspirin nonresponders is 5.5-45% in patients with various cardiovascular diseases. Those who have aspirin nonresponders have a greater risk of clinically cardiovascular events. The purpose of the study was to look for the prevalence, associated factors and the outcomes of aspirin nonresponders among patients with ischaemic stroke. Patients with ischaemic stroke who were treated during January 2011-August 2011 were included. Urine 11-dehydro-thromboxane B2 (dTXB2) was measured to determine the response to aspirin in patients. The demographics and vascular risk factors were compared between patients who were classified as aspirin responders or aspirin nonresponders. The outcomes of the study were favourable outcome, cardiovascular events and mortality. There were 182 patients included during the study period: 128 patients with an acute ischaemic stroke and 54 patients with a stable ischaemic stroke. Ninety patients (49.5%) were found to be aspirin nonresponders. Multivariate analysis revealed that stroke presentation (acute stroke) was the only factor associated with aspirin nonresponders [odds ratio (OR) 2.38, 95% confidence interval (CI) 1.193-4.746, P = 0.014]. With a mean follow-up time of 16 months, aspirin nonresponders had a less favourable outcome (54 vs. 83%, OR 0.24; 95% CI 0.11-0.51, P < 0.001), marginally higher cardiovascular events (11 vs. 2%, OR 4.48; 95% CI 0.92-21.37, P = 0.045) and higher mortality (12 vs. 1%, OR 10.52; 95% CI 1.3-85.28, P = 0.007). The prevalence of aspirin nonresponders was rather high in Thai patients with ischaemic stroke. Aspirin nonresponders had a less favourable outcome, higher cardiovascular events and death rate.

Effects of different doses, enteric-coated preparation of aspirin, and sex on urinary 11-dehydrothromboxane B2 in healthy volunteers.

There is scarce information about the effects of different doses and enteric-coated preparation of aspirin on platelet function, especially in Asian people, evaluated by the measurement of urinary 11-dehydrothromboxane B2 (dTXB2). The objective of the present study was to assess the effects of different doses, enteric-coated preparation of aspirin, sex and also the effects of timing of urine collection on urinary dTXB2 level in healthy volunteers. Thirty healthy volunteers were included. Each volunteer took three preparations of aspirin (aspirin 81 mg, enteric-coated aspirin 300 mg and aspirin 300 mg) for 7 days. Urine dTXB2 level was measured at baseline, day 3, and day 7 after taking each preparation of aspirin. There was no significant difference in the effects of different doses of aspirin (81 vs. 300 mg, 50.7 vs. 61.8 ng/mmol creatinine, P = 0.248), preparations (enteric-coated vs. nonenteric-coated aspirin, 61.8 vs. 67.9 ng/mmol creatinine, P = 0.527) and time of urine collection (day 3 vs. day 7, 51.7 vs. 49.9 ng/mmol creatinine, P = 0.448). Female volunteers showed a trend to have higher urinary dTXB2 than male volunteers at baseline and after taking aspirin. This study showed no significant difference in urinary dTXB2 level after taking different doses and enteric-coated preparation of aspirin in healthy volunteers.