Báo cáo khoa học: MicroRNA-143 reduces viability and increases sensitivity to 5-fluorouracil in HCT116 human colorectal cancer cells potx

MicroRNA-143 reduces viability and increases sensitivity to 5-fluorouracil in HCT116 human colorectal cancer cells Pedro M Borralho1, Betsy T Kren2, Rui E Castro1, Isabel B Moreira da Silva1, Clifford J Steer2,3 ´ and Cecılia M P Rodrigues1 Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Portugal Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN, USA Keywords 5-fluorouracil; apoptosis; chemosensitizer; ERK5; miR-143 Correspondence C M P Rodrigues, Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal Fax: +351 21 794 6491 Tel: +351 21 794 6400 E-mail: cmprodrigues@ff.ul.pt (Received 29 April 2009, revised August 2009, accepted 15 September 2009) doi:10.1111/j.1742-4658.2009.07383.x MicroRNAs are aberrantly expressed in cancer; microRNA-143 (miR-143) is down-regulated in colon cancer HCT116 human colorectal cancer cells were used to investigate the biological role of miR-143 Transient miR143 overexpression resulted in an approximate 60% reduction in cell viability In addition, stable miR-143 overexpressing cells were selected with G418 and exposed to 5-fluorouracil Increased stable expression of miR143 was associated with decreased viability and increased cell death after exposure to 5-fluorouracil These changes were associated with increased nuclear fragmentation and caspase -3, -8 and -9 activities In addition, extracellular-regulated protein kinase 5, nuclear factor-jB and Bcl-2 protein expression was down-regulated by miR-143, and further reduced by exposure to 5-fluorouracil In conclusion, miR-143 modulates the expression of key proteins involved in the regulation of cell proliferation, death and chemotherapy response In addition, miR-143 increases the sensitivity of colon cancer cells to 5-fluorouracil, probably acting through extracellular-regulated protein kinase ⁄ nuclear factor-jB regulated pathways Collectively, the data obtained in the present study suggest anti-proliferative, chemosensitizer and putative pro-apoptotic roles for miR-143 in colon cancer Introduction MicroRNAs (miRNAs) are a recently discovered and growing class of small noncoding, single-stranded RNAs that negatively regulate gene expression miRNA biogenesis initiates by RNA polymerase II transcription of a primary transcript (pri-miRNA) This pri-miRNA is processed in the nucleus by the RNase III enzyme Drosha, producing pre-miRNAs, comprising hairpin structures of approximately 70 nucleotides Subsequently, pre-miRNAs are exported to the cytoplasm through an Exportin-5-dependent mechanism, and further processed by the RNAse III enzyme Dicer, producing miRNA duplexes One strand of the duplex is incorporated into the RNA-induced silencing complex, whereas the other is usually rapidly degraded In mammals, miRNA-directed gene silencing occurs primarily via incomplete miRNA binding to 3¢UTRs of target mRNAs, which represses translation and, in some cases, leads to mRNA degradation [1] More than 670 miRNAs have been identified in humans, but mRNA targets and regulatory pathways Abbreviations ERK5, extracellular-regulated protein kinase 5; 5-FU, 5-fluorouracil; LDH, lactate dehydrogenase; miRNA, microRNA; miR-143, miRNA-143; miR-145, miRNA-145; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NF, nuclear factor; pNA, p-nitroanilide FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6689 miR-143 modulates 5-FU cytotoxicity P M Borralho et al have only been explored for a handful of miRNAs Furthermore, miRNAs are significantly involved in the regulation of a myriad of biological processes, such as differentiation, proliferation and apoptosis [2], which are commonly altered in cancer It is now apparent that miRNAs are differentially expressed in a wide array of human cancers, including colorectal cancer [3,4] Nevertheless, the molecular signalling pathways modulated by miRNAs, which play a role in colorectal cancer, as well as cancer in general, are only partially characterized miRNA-143 and miRNA-145 (miR-143 and miR-145) expression was found reduced in tumours versus matched normal mucosa, both at the adenomatous and cancer stages of colorectal neoplasia, as well as in colon cancer cell lines [3,5] Furthermore, miR-143 is a putative cancer biomarker that is also down-regulated in B-cell malignancies [6], bladder [7] and cervical cancer [8] miR-143 targets extracellularregulated protein kinase (ERK5) during adipocyte differentiation [9] and in DLD-1 colon cancer cells [5] To date, miR-143 has been demonstrated to directly target the mRNA of KRAS [10,11], ERK5 [12], DNMT3A [13] and ELK1 [14] Despite growing evidence for an anti-proliferative and putative pro-apoptotic role for miR-143, a more detailed understanding of miR-143 biological function is warranted because reduced miR-143 expression may deregulate molecular signalling pathways with direct implications in cancer onset and ⁄ or progression and response to chemotherapy ERK5 is a mitogen-activated protein kinase that is regulated by a wide range of mitogens and cellular stresses In addition, activated ERK5 is involved in cell survival, differentiation and proliferation Several ERK5 targets and potential regulators of cell proliferation have been identified, including c-Myc, cyclin D1 and nuclear factor (NF)-kB [15] ERK5 activation of NF-jB promotes cellular transformation [16] and is a critical factor for G2–M cell cycle progression and timely mitotic entry [17] NF-jB is a critical transcription factor involved in the suppression of apoptosis, stimulation of cell growth and the modulation of genes that appear to be important in tumour promotion, angiogenesis and metastasis NF-jB activation is also associated with increased resistance to chemotherapeutic agents [18] Therefore, strategies aimed at reducing ERK5 and NF-jB signalling may modulate tumour growth and sensitivity to chemotherapeutic agents 5-Fluorouracil (5-FU) has been the drug of choice for the treatment of colorectal cancer for more than four decades Its limited efficacy as a single agent for advanced colorectal cancer has been improved by combination with newer chemotherapeutic agents [19] 6690 5-FU has been shown to induce apoptosis in colon cancer cell lines [20,21] The ability of tumour cells to evade apoptosis is an enormous obstacle for effective treatment Consequently, strategies aiming to overcome tumour cell resistance to chemotherapy and to increase drug efficacy, thereby minimizing toxic effects, are critically important The molecular mechanisms of 5-FU cytotoxicity have been characterized, especially its ability to incorporate into RNA and DNA and to inhibit thymidylate synthase 5-FU has recently been shown to modulate miRNA expression in colon cancer cells [22] However, the relationship between 5-FU and miRNAs, their potential interactions and their relevance for drug efficacy have not been extensively studied Nevertheless, drug function could potentially be improved via modulation of miRNAs that play a role in chemoresistance In the present study, we evaluated the role of miR-143 in the response of HCT116 colon cancer cells to 5-FU The results obtained demonstrate that miR-143 decreased colon cancer cell viability and increased 5-FU sensitivity, suggesting that it may act as a potential chemosensitizer to 5-FU in colon cancer cells In addition, miR-143 overexpression resulted in the down-regulation of ERK5, NF-jB and Bcl-2 protein expression, which was further reduced by 5-FU Collectively, our data indicate that reduced miR-143 expression in colon cancer may contribute to unchecked proliferation and decreased sensitivity to 5-FU Furthermore, increasing miR-143 expression in colon cancer cells may comprise a promising strategy for reducing tumour growth and aggressiveness, at the same time as increasing sensitivity to 5-FU Results miR-143 overexpression reduces colon cancer cell viability We first evaluated miR-143 expression in colon cancer cells lines by semi-quantitative RT-PCR, using total RNA and miRNA-enriched RNA (providing a higher abundance of < 200 nucleotides RNA molecules per lg RNA) The results obtained confirmed that miR-143 levels were almost undetectable in HCT116, LoVo, SW480 and SW620 colon cancer cells, using up to 500 ng of miRNA-enriched RNA and 30 PCR cycles (data not shown) By contrast, miR-143 expression was readily detected from total heart RNA, using lower RNA input, whithout miRNA-enrichement This was achieved with as little as 25 ng of total heart RNA, and at a lower PCR cycle number, thus reinforcing the notion that mir-143 is expressed at low FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS P M Borralho et al miR-143 modulates 5-FU cytotoxicity levels in colon cancer cells U6 was used throughout as normalization control HCT116 cells were chosen for further experiments because the molecular pathways of 5-FU cytotoxicity have already been explored in this cell line [20] We next confirmed that mature miR-143 is produced from pCR3-pri-miR-143 vector and that it specifically binds to miR-143 sensor vector This was achieved by co-transfecting HCT116 cells with pCR3-pri-miR-143, a firefly luciferase miR-143 sensor plasmid (miR-143 sensor) as a reporter for mature miR-143 expression, and with either miR-143 specific inhibitor (anti-miR143) or control (anti-miR-control) pRL-SV40 was also co-transfected and used as a normalization control The results obtained showed that the lower availability of mature miR-143 after anti-miR-143 co-transfection led to increased firefly activity (Fig 1A) After validating the vectors, we determined the effect of miR-143 transient overexpression in HCT116 colon cancer cells The results obtained demonstrated that pri-miR-143 overexpression reduced HCT116 cell viability by approximately 60%, at 48 h post-transfection (P < 0.05) as evaluated by the 3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium (MTS) assay (Fig 1B) In addition, miR-143 overexpression increased cell death as early as 24 h post-transfection (data not shown) pCR3-empty transfection resulted in no significant change in cell viability A B miR-143 increases 5-FU cytotoxicity We next determined the effects of 5-FU on colon cancer cells with endogenous low levels of miR-143 Therefore, we exposed HCT116 colon cancer cells to lm 5-FU, a clinically relevant concentration [23] that has been shown to induce cell death and apoptosis [20] 5-FU reduced cell viability by approximately 40% and increased general cell death by approximately 60% after 48 h of exposure (P < 0.01) After miR-143 transient overexpression, 5-FU also reduced cell viability and increased cell death compared to pCR3-empty 5-FU exposed cells (data not shown) This prompted us to create a stable miR-143 overexpression model by transfecting HCT116 cells with pCR3-pri-miR-143 and pCR3-empty and selecting with G418 On the basis of miR-143 expression levels evaluated by luciferase activity after pGL3-miR-143 sensor and pGL3-control, we selected a miR-143 overexpression clone and an empty vector clone, designated HCT116OV3 and HCT116-EM1, respectively (Fig 2A) In addition, we measured miR-143 expression by TaqMan real-time PCR, using specific primers for mature Fig Mature miR-143 overexpression decreases HCT116 cell viability Cells were transfected with the indicated plasmids and 50 or 100 nM anti-miR inhibitors, and analyzed at 48 h post-transfection (A) Cells were lysed and firefly and renilla luciferase activities were determined by the dual luciferase assay (B) Cell viability was evaluated by MTS metabolism assays Cells were then lysed and renilla luciferase activity was determined by the dual luciferase assay, for normalization of the MTS metabolism assay The results are expressed as the mean ± SEM from at least three independent experiments  P < 0.01 and *P < 0.001 compared to controls; §P < 0.05 compared to pCR3-empty + pRL-SV40 miR-143, and RNU6B for normalization The results obtained showed that miR-143 expression is increased five-fold in HCT116-OV3 cells compared to HCT116 parental cells (P < 0.01) (Fig 2B) We also evaluated whether 5-FU directly modulates the expression of mature miR-143 Total RNA was extracted from HCT116, HCT116-OV3 and HCT116EM1 cells exposed to lm 5-FU for 48 h Mature FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6691 miR-143 modulates 5-FU cytotoxicity A P M Borralho et al B C Fig 5-FU increases miR-143 expression in HCT116 cells (A) miR-143 expression by luciferase activity assays Cells were co-transfected with either pGL3-miR-143 sensor or pGL3-control and pRL-SV40 and analyzed at 48 h after transfection Cells were lysed and luciferase activity was evaluated with the dual luciferase assay miR-143 levels were expressed as the luciferase signal ratio of pGL3-miR-143 sensor to pGL3-control cells (B, C) Cells were harvested for total RNA extraction after 48 h in culture with and without lM 5-FU exposure miR-143 expression was evaluated from 1.33 lL cDNA of 10 ng of total RNA RT reactions, using specific primers for miR-143 and RNU6B for normalization miR-143 expression levels were calculated by the DDCt method, using HCT116 control cells as calibrator The results are expressed as the mean ± SEM fold-change compared to HCT116 cells from three to six independent experiments *P < 0.001 and  P < 0.05 compared to controls miR-143 expression was measured by TaqMan realtime PCR Interestingly, the results obtained showed that miR-143 increased almost three-fold in parental HCT116 and HCT116-EM1 cells, but by only two-fold in HCT116-OV3 (P < 0.05) (Fig 2C) We next evaluated the effect of increased mature miR-143 expression in colon cancer cells exposed to 5-FU For this purpose, cells were exposed to 1–10 lm 5-FU for 72 h, and cell viability and cell death were 6692 evaluated by the MTS metabolism and lactate dehydrogenase (LDH) release assays The results obtained indicated that mature miR-143 enhanced sensitivity to 5-FU Indeed, cell viability was reduced and cell death was increased in HCT116-OV3 compared to parental and HCT116-EM1 cells, after 1–10 lm 5-FU exposure for 72 h (P < 0.05) (Fig 3A) In addition, increased sensitivity to 5-FU was also observed in G418 selected pCR3-pri-miR-143 transfected mixed populations compared to pCR3-empty transfected mixed populations after exposure to same doses of 5-FU (Fig 3B) Furthermore, 5-FU-induced apoptosis was enhanced in HCT116-OV3 cells, resulting in increased caspase-3, -8 and -9 activities and subsequent nuclear fragmentation compared to parental HCT116 and HCT116-EM1 control cells (P < 0.05) (Fig 4) Finally, we evaluated miR-143 expression in SW480 cancer cells exposed to 5-FU SW480 cells were less sensitive to 5-FU than HCT116 cells (Fig 5A) In addition, 5-FU up-regulated miR-143 expression in both cell lines (Fig 5B) Nevertheless, miR-143 expression was significantly lower in SW480 cells The DCt values were 12.10 ± 0.35 in SW480 cells compared to 5.45 ± 0.08 in HCT116 cells (P < 0.001), whereas RNU6B Ct values for control and 5-FU-treated HCT116 and SW480 cells were 24.43 ± 0.22 and 24.42 ± 0.25, respectively (P = 0.954) Collectively, this represents an increase of almost 100-fold in miR-143 expression in HCT116 relative to SW480 cells, thereby reinforcing the potential involvement of miR-143 in 5-FU sensitivity miR-143 and 5-FU down-regulate ERK5, NF-jB and Bcl-2 protein expression ERK5 targeting by miR-143 has been shown in adipocytes [9] in addition to DLD-1 colon cancer cells [5] More recently, miR-143 has been demonstrated to directly target the mRNA of KRAS [10,11], ERK5 [12], DNMT3A [13] and ELK1 [14] To determine whether miR-143 modulates ERK5 in HCT116 cells as well as its relevance to 5-FU cytotoxicity, we evaluated ERK5 protein expression after transfection of miR-143 precursor molecules (pre-miR-143) and a nonspecific control (pre-miR-control) The results obtained showed that miR-143 reduced ERK5 protein expression in a dose-dependent manner, leading to an approximate 70% reduction at 48 h post-transfection with 80 nm pre-miR-143 (P < 0.001) (Fig 6A, upper panel) In addition, after transfection with 80 nm pre-miR-143, 5-FU further reduced ERK5 protein expression in a time-dependent manner, reaching a reduction of 50%, 60% and 80% at 24, 48 and 72 h, respectively FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS P M Borralho et al miR-143 modulates 5-FU cytotoxicity A B Fig mir-143 overexpressing cells are more sensitive to 5-FU cytotoxicity Cells were exposed to either 1–10 lM 5-FU or no addition (control) for 72 h Mixed populations of miR-143 overexpressing cells (pCR3-pri-miR-143 mix) and respective control cells (pCR3-empty mix) were generated by transfecting HCT116 cells with pCR3-pri-miR-143 or pCR3-empty vector, respectively Cells were selected and maintained with mgỈmL)1 G418, under the same conditions as those used for single-clone derived HCT116-OV3 and HCT116-EM1 cells (A, B) Cells with higher miR-143 expression (HCT116-OV3 and pCR3-pri-miR-143 mix) displayed decreased viability and increased cell death after exposure to 5-FU Cell viability and cell death were evaluated by MTS metabolism and LDH activity assays, respectively In the far right panels of (A) and (B), LDH normalized to MTS is also plotted for the mean values of each 5-FU concentration used The results are expressed as the mean ± SEM fold-change compared to controls from at least three independent experiments *P < 0.01 and  P < 0.05 from HCT116-EM1 No significant changes were observed between HCT116 and HCT116-EM1 or between HCT116 and pCR3-empty mix for 1–10 lM 5-FU (P < 0.05) compared to controls (Fig 6A, lower panel) In addition, we were also able to regulate ERK5 protein expression by modulating the availability of mature miR-143 (Fig 6B) At 72 h after co-transfection of 40 nm pre-miR-143 with 60 nm of its specific inhibitor anti-miR-143, ERK5 was increased compared to pre-miR-143 and anti-miR control co-transfection experiments (Fig 6B, lane versus 5) Moreover, pre-miR-control and anti-miR-143 co-transfection also increased ERK5 protein expression compared to co-transfection of pre-miR-control and anti-miR-control (Fig 6B, lane versus 7) Exposure to 5-FU resulted in a further reduction of ERK5 protein expression (Fig 6B, lanes 9–14 versus 1–7) Accordingly, higher miR-143 abundance decreased cell viability (Fig 6C) Finally, ERK5 expression was knocked-down in HCT116 cells by transfecting 80 nm of specific ERK5 siRNA and then exposing cells to 5-FU The results obtained show that ERK5 silencing augmented apoptosis compared to mock-transfected cells, whereas increasing 5-FU-induced apoptosis (P < 0.05) (Fig 6D) Furthermore, we also found that stable miR-143 overexpressing cells express lower levels of ERK5, NF-jB and Bcl-2 proteins compared to control and parental HCT116 cells Furthermore, 5-FU potentiated ERK5, NF-jB and Bcl-2 expression knockdown (Fig 7) These results further indicate that miR-143 is a key player in the regulation of cell proliferation and the response to 5-FU growth inhibition ⁄ cytotoxicity in HCT116 cells, probably by acting through the ERK5 ⁄ NF-jB axis Discussion It is now well-established that miRNAs regulate a plethora of crucial cellular functions, including cell growth, differentiation and apoptosis, which are commonly altered in cancer cells In the present study, we evaluated the effect of miR-143 overexpression on HCT116 colon cancer cells HCT116 cells were transfected with pCR3-pri-miR-143, and the respective control, pCR3-empty miR-143 production was assessed by luciferase assays The results obtained demonstrate FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6693 miR-143 modulates 5-FU cytotoxicity P M Borralho et al Fig HCT116-OV3 cells are more sensitive to 5-FU-induced apoptosis Cells were exposed to lM 5-FU and processed for evaluation of nuclear morphology after Hoechst staining at 24 h, and for caspase activity assays at 12 h The results are expressed as the mean ± SEM fold-change compared to controls from at least three independent experiments *P < 0.01 and  P < 0.05 from HCT116-EM1 No significant changes were observed between HCT116 and HCT116-EM1 that increased pri-miR-143 expression reduced cell viability in pCR3-pri-miR-143 transfected cells by 60% compared to control cells These results are in accordance with a previous study, in which pre-miR-143 transfection in SW480 and DLD-1 colon cancer cells reduced cell viability in a dose-dependent manner [5] We were particularly interested in investigating the effect of miR-143 on the cellular response to 5-FU 5-FU is a well-known apoptosis-inducing drug; indeed, it was shown previously that 5-FU induces apoptosis in HCT116 cells [20] 5-FU has been used in the clinic for several decades, and its metabolic pathways and cytotoxic modes of action through the inhibition of thymidylate synthase activity and incorporation into RNA and DNA are well known However, the molecular pathways modulated by 5-FU that lead to cell growth inhibition and cell death induction are not entirely understood, nor are the mechanisms of tumour cell escape from 5-FU cytotoxicity In addition, little is known about the role of miRNAs in modulating the tumour cell response to chemotherapeutic agents, such as 5-FU Nevertheless, 5-FU alters the expression of a set of 22 miRNAs in colon cancer cell lines [22] Furthermore, S-1, a fourth-generation 5-FU-based oral drug developed to improve efficacy, also alters the expression levels of certain miRNAs, as demonstrated in tumour tissue from patients undergoing S-1 therapy [24] 6694 To evaluate the effect of miR-143 on 5-FU sensitivity, we created stable miR-143 overexpressing cells (HCT116-OV3) and the respective control cells (HCT116-EM1) by pCR3-pri-miR-143 and pCR3empty transfection, respectively, and G418 selection miR-143 expression was evaluated by luciferase assays and also by TaqMan real-time PCR with specific primers for miR-143 and RNU6B The results obtained showed that miR-143 expression was increased five-fold in HCT116-OV3 cells Cells were then exposed to different concentrations of 5-FU for 72 h to plot growth inhibition and cell death dose–response curves 5-FU at a concentration 1–10 lm was significantly more cytotoxic in HCT116-OV3 cells compared to HCT116-EM1 control or HCT116 parental cells These effects were already evident 48 h after 5-FU exposure but more intense after 72 h of drug exposure Furthermore, we also observed higher sensitivity to 5-FU in mixed populations of miR143 overexpressing cells compared to controls Curiously, a chemosensitizer role of miR-143 to 5-FU has also been reported in gastric cancer cells Indeed, transfection of pre-miR-143 into MKN-1 cells was shown to reduce the number of viable cells after exposure to 10 lm 5-FU [25] Collectively, these data indicate that miR-143 is an important modulator of 5-FU sensitivity not only in colon cancer, but also in other types of cancer, particularly those of the gastrointestinal tract FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS P M Borralho et al A B Fig SW480 cells are less sensitive to 5-FU than HCT116 cells and express less miR-143 Cells were exposed to either 1–100 lM 5-FU or no addition (control) for 72 h (A) HCT116 cells displayed increased growth inhibition and cell death after exposure to 5-FU, as evaluated by MTS metabolism and LDH activity assays, respectively (B) Cells were exposed to lM 5-FU for 72 h and miRNAenriched RNA was extracted with a mirVanaÔ PARIS kit miR-143 expression was evaluated from lL of cDNA of a 50 ng miRNAenriched RNA RT reaction, using specific primers for miR-143, and RNU6B for normalization miR-143 expression levels were calculated by the DDCt method, using control cells as calibrator miR-143 was up-regulated in HCT116 and SW480 cells after 5-FU exposure by approximately three- and two-fold, respectively RNU6B Ct values for control and 5-FU-treated HCT116 and SW480 cells were 24.43 ± 0.22 and 24.42 ± 0.25, respectively (P = 0.954) The results are expressed as the mean ± SEM percentage of growth inhibition, LDH activity or miR-143 expression from at least three independent experiments (A) *P < 0.01 and  P < 0.05 from SW480 cells; (B) *P < 0.001 compared to HCT116 and  P < 0.05 compared to the respective nontreated cells We next selected lm 5-FU to further explore the differential sensitivity of our cell model We have previously shown that it efficiently triggers apoptosis in HCT116 cells [20] Furthermore, the results obtained in the present study now show that lm 5-FU induced growth inhibition by 55% in addition to a two-fold increase in cell death Notably, 5-FU significantly increased caspase activation and nuclear fragmentation in HCT116-OV3 compared to control and parental cells We have also evaluated miR-143 expression after 48 h of 5-FU exposure and found it to be significantly increased Curiously, 5-FU increased miR-143 modulates 5-FU cytotoxicity miR-143 expression to a lesser extent in HCT116-OV3 than in HCT116-EM1 and HCT116 cells, which may be attributed to the relatively high levels of miR-143 in HCT116-OV3 cells Furthermore, up-regulation of miR-143 by 5-FU also occurs in SW480 cells that are less sensitive to 5-FU cytotoxicity However, the basal expression of miR-143 is significantly lower in SW480 cells than in HCT116, which underlines the relevance of miR-143 expression on 5-FU sensitivity The results obtained in the present study are in agreement with a recent report showing that p53 enhances the post-transcriptional maturation of miR-143 in response to DNA damage [10] Using wild-type p53 HCT116 cells exposed to the potent p53 inducer doxorubicin, and despite no significant changes in pri-miR-143 levels, there was increased processing of pri-miR-143 into preand mature miR-143 [10] In addition, this DNA-damage-induced up-regulation of pre-miR-143 and mature miR-143 was diminished in p53 null HCT116 cells, thereby suggesting that increased mature miR-143 expression may be a result of increased p53 expression These data may explain the increased levels of mature miR-143 that we found in the present study after exposure of wild-type p53 HCT116 cells to 5-FU because it has previously been shown that 5-FU also strongly increases p53 expression in this cell line [20] Interestingly, when mutant p53 (R273H) was introduced into p53 null HCT116 cells, there was a reduction in pre- and mature miR-143 production, contrasting with constant levels of pri-miR-143 [10], which suggests that mutant p53 hampers miR-143 processing in a transcription-independent manner These results are once again in agreement with the results obtained in SW480 cells In these cells expressing the R273H mutant p53, we observed a dramatic approximately 100-fold decrease in mature miR-143 expression compared to wild-type p53 HCT116 cells However, in SW480 cells, we also found an increase in mature miR-143 after 5-FU exposure, which may indicate that there is some mature miR-143 production that escapes wild-type p53 control Nevertheless, the levels of mature miR-143 in SW480 cells treated with 5-FU are much lower than those observed in untreated HCT116 cells This finding may contribute to or be associated with SW480 lower sensitivity to 5-FU cytotoxicity We cannot conclude whether the basal difference in miR-143 expression between HCT116 and SW480 cells results from loss of transcriptional or post-transcriptional p53 modulation p53 has been shown to increase mature miR-145 expression and bind to a putative response element located upstream of miR-145 [26] In addition, miR-143 and miR-145 are highly conserved miRNAs in close genomic proximity [14,27], suggesting FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6695 miR-143 modulates 5-FU cytotoxicity P M Borralho et al A C D B Fig miR-143 and 5-FU down-regulate ERK5 protein expression (A) HCT116 cells were transfected with 40 or 80 nM pre-miR-143 or premiR-control and ERK5 protein expression was evaluated at 48 h after transfection (upper panel) HCT116 cells were transfected with 80 nM pre-miR-143 or pre-miR-control At h after transfection, cells were exposed to lM 5-FU for 24, 48 or 72 h ERK5 protein expression was evaluated at the indicated time-points (lower panel) (B) HCT116 cells were transfected with 40 nM pre-miR-143 or pre-miR-control, or with additional 60 nM anti-miR-control or anti-miR-143 At h after transfection, cells were exposed to lM 5-FU for 72 h, at which time ERK5 protein expression was evaluated (C) HCT116 cells were transfected with 80 nM pre-miR-143, pre-miR-control, anti-miR-143 or anti-miR-control At h after transfection, cells were exposed to lM 5-FU for 72 h, at which time cell viability was determined The results are expressed as the fold-change of LDH normalized to MTS for pre-miR-143 and anti-miR-143 compared to the respective controls Black bar, ratio of pre-miR-143 over pre-miR-control; white bar, ratio of anti-miR-143 over anti-miR-control (D) HCT116 cells were transfected with 80 nM ERK5 siRNA or mock transfected Twenty-four hours after transfection, cells were exposed to lM 5-FU for 48 h and processed for evaluation of nuclear morphology after Hoechst staining ERK5 protein expression was also evaluated at the same time The results are expressed as the mean ± SEM fold-change compared to controls from at least three independent experiments Representative blots from at least three independent experiments are shown *P < 0.05 compared to control HCT116 cells (mock) and  P < 0.05 compared to 5-FU-treated HCT116 cells (5-FU-treated mock) that their expression is linked to a bicistronic primary precursor Indeed, the use of specific primers for miR-143 and miR-145 resulted in amplicons containing both miR-143 and miR-145, suggesting that similar mechanisms are responsibe for the regulation of miR-143 and miR-145 [14] Nevertheless, p53 induction by doxorubicin increases the transcriptional activation of miR-34a, which is a known p53 transcriptional target, and does not increase pri-miR-143 or pri-miR-145 [10] In addition, the increased maturation of miR-145 after DNA damage was similar to the reported increase in miR-143 maturation in multiple cell lines Further studies are required to clarify the molecular details of the relationship between p53 and miR-143 and miR-145 6696 miR-143 was initially shown to regulate adipocyte differentiation by targeting ERK5 [9] Recently, ERK5 [5], KRAS [10,11] and DNMT3A [13] were also shown to be negatively modulated by miR-143 in colon cancer cells More importantly, these genes have been proven to be direct targets of miR-143 [10–13] Our data obtained in HCT116 cells are in accordance with previous reports and show that miR-143 overexpression reduces ERK5 protein expression ERK5 is involved in cell proliferation and differentiation [28], as well as the inhibition of endothelial cell apoptosis [29] In addition, ERK5 has been identified as a pro-survival kinase during mitosis, whereas ERK5 knockdown by RNA interference induced apoptosis [30] ERK5 is also a critical factor FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS P M Borralho et al Fig miR-143 and 5-FU down-regulate ERK5, NF-jB and Bcl-2 protein expression Populations of HCT116 cells, miR-143 overexpressing cells (HCT116-OV3 and pCR3-pri-miR-143 mix) and respective control cells (HCT116-EM1 and pCR3-empty mix) were synchronized to early S-phase by a double thymidine block Eighthours after seeding, mM thymidine was added and cells were cultured for 14 h Cells were then released from the block for 10 h in media lacking thymidine, followed by an additional 14 h of culture in the presence of mM thymidine (second block) Cells were released from the second thymidine block into media with 5-FU or no addition (control) and harvested for protein extraction 72 h later Representative blots from at least three independent experiments are shown àP < 0.05 compared to HCT116;  P < 0.05 compared to 5-FU-treated HCT116 cells; §P < 0.05 compared to the respective untreated cells; *P < 0.05 compared to HCT116-EM1 or pCR3-empty mix cells; #P < 0.05 compared to 5-FU-treated HCT116-EM1 or 5-FU-treated pCR3-empty mix in G2–M cell cycle progression and timely mitotic entry because stimulation of ERK5 activated NF-jB, whereas inhibition of NF-jB at G2–M significantly delayed mitotic entry [17] These results suggest a potential crosstalk between ERK5 and the apoptotic machinery The results obtained in the present study are in agreement with this notion by demonstrating that miR-143 overexpression leads to a significant reduction of ERK5 and NF-jB protein expression Because ERK5 directly activates NF-jB to promote cell cycle progression through G2–M, reduced levels of these proteins may account for reduced cell growth miR-143 modulates 5-FU cytotoxicity as well as an increased response to 5-FU In addition, we also see a strong down-regulation of the antiapoptotic protein Bcl-2 associated with higher miR-143 expression, which may contribute to the putative pro-apoptotic role of this miRNA Our results are particularly relevant because they suggest that miR-143 is an endogenous cell growth ⁄ proliferation master switch, in which any loss may result in unchecked cellular proliferation via ERK5 ⁄ NF-jB signalling In turn, this may constitute a key event for colon cancer onset and progression miR-143 expression was evaluated in situ in mouse colonic epithelial crypts [27] and found mainly in the cytoplasm, where it was more intense in the ascending more differentiated pre-apoptotic glandular cells than in the immature, proliferating cells at the bottom of crypts Therefore, the extremely low miR-143 expression levels in cancer and, in particular in colon cancer, may be an important factor contributing to tumour growth and ⁄ or escape from apoptosis In addition, miR-143 expression has been shown to increase after a-mangostin exposure in human colon cancer DLD-1 cells, resulting in increased apoptosis [31] Furthermore, a-mangostin acted synergistically with low dose 5-FU, increasing DLD-1 growth inhibition [31] Our data also show that 5-FU increases miR-143 expression, which may potentiate 5-FU sensitivity, suggesting a feed-forward mechanism of action The results obtained in the present study provide additional insight into miR-143 regulated pathways and their impact on 5-FU sensitivity Exposure of miR-143 overexpressing cells to 5-FU potentiated a significant decrease in ERK5, NF-jB and Bcl-2 protein expression and resulted in increased 5-FU cytotoxicity Collectively, our findings emphasize the potential pivotal relevance of miR-143 in the colon cancer environment and suggest that it has a role as chemosensitizer to 5-FU Further studies are necessary to elucidate the full extent of the molecular signalling pathways and players modulated by miR-143 Materials and methods Cell culture HCT116 cells were grown in DMEM supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA), 1% l-glutamine 200 mm (Merck and Co Inc., Whitehouse Station, NJ, USA) and 1% antibiotic ⁄ antimycotic solution (Sigma Chemical Co., St Louis, MO, USA) and maintained at 37 °C in a humidified atmosphere of 5% CO2 In selected experiments, HCT116 cells were compared with SW480, LoVo and SW620 human colorectal cancer FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6697 miR-143 modulates 5-FU cytotoxicity P M Borralho et al cells All cell lines were grown under identical conditions Cells were seeded at 0.75 · 105 cellsỈmL)1 for pre-miR-143 tranfections; · 105 cellsỈmL)1 for morphological assessment of apoptosis; and at · 105 cellsỈmL)1 for RNA and protein extraction, cell viability and cell death assays Cell synchronization Cell synchronization was performed using a double thymidine block (early S-phase) Eight-hours after seeding 0.75 · 105 cellsỈmL)1, mm thymidine (Sigma) was added and cells were cultured for 14 h Cells were then released from block for 10 h in media lacking thymidine, followed by an additional 14 h culture in the presence of mm thymidine (second block) Cells were released from second thymidine block into media with either 5-FU or no addition (control) Cells were harvested for protein extraction at the indicated times Generation of HCT116 cells with stable expression of miR-143 HCT116 cells were selected and maintained with mgỈmL)1 G418 (Invitrogen), after transfection with pCR3-pri-miR143 and pCR3-empty vectors Isolated single clone-derived cell foci were picked up by ring cloning strategies, expanded and propagated in complete media plus G418 In addition, mixed populations of miR-143 overexpressing and control cells were obtained in a similar manner, without the singlecell selection step miR-143 expression was evaluated by luciferase assay after co-transfection and normalization with pGL3-miR-143 sensor and pRL-SV40 This assay was performed regularly to control cell clone consistency In addition, TaqMan real-time PCR (Applied Biosystems) was used to confirm the expression of mature miR-143 in cell clones Evaluation of cell death and viability 5-FU exposure 5-FU (Sigma) stock solutions of 100 and mm were prepared in dimethyl sulfoxide Twenty-four hours after plating, cells were incubated with either 5-FU or no addition (control) for the indicated times For the 5-FU dose– response curves, media was removed 24 h after plating and replaced with fresh media containing 5-FU The final dimethyl sulfoxide concentration was always 0.1% Transfection of miR-143 vectors, anti-miR-143 inhibitor, pre-miR-143 and ERK5 siRNA HCT116 cells were transiently transfected with miR-143 overexpression vector coding for the miR-143 precursor (pCR3-pri-miR-143) and miR-143 sensor comprising two sequences complementary to mature miR-143 sequence (pGL3-miR-143 sensor) [9] pRL-SV40 (Promega, Madison, WI, USA) was used for transfection normalization pGL3control plasmid (Promega) and pCR3-empty vector were used as negative controls To further validate the experimental model, cells were co-transfected with anti-miR inhibitors by adding either 50 or 100 nm anti-miR-143 or anti-miR-control inhibitors to the vector mixture described above In addition, HCT116 cells were transfected with pre-miR-143, pre-miR-control, anti-miR-143 and anti-miRcontrol at a final concentration of 40 or 80 nm We also co-transfected HCT116 cells with 40 nm pre-miR-143 or pre-miR-control plus 60 nm anti-miR-143 or anti-miRcontrol, at a final concentration of 100 nm ERK5 silencing was performed using 80 nm MAPK7 SilencerÒ Select Pre-Designed & Validated siRNA (all from Applied Biosystems, Foster City, CA, USA) Transfections were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions 6698 At the indicated times, general cell death was evaluated by the LDH assay kit (Sigma) LDH activity was evaluated in cell culture media, using a Bio-Rad microplate reader Model 680 (Bio-Rad, Hercules, CA, USA) Cell viability was evaluated with CellTiter96Ò AQueous Non-Radioactive Cell Proliferation Assay (Promega), using MTS inner salt Finally, cells were processed for luciferase assay and transfection efficiency normalization Luciferase activity At the indicated times, firefly and renilla luciferase activities were measured using the Dual-LuciferaseÒ Reporter Assay System (Promega) Renilla luciferase activity was used as a transfection normalization control Nuclear morphology Hoechst labelling of cells was used to detect apoptotic nuclei Attached cells were fixed with 4% paraformaldehyde in NaCl ⁄ Pi (pH 7.4) and incubated with Hoechst dye 33258 (Sigma Chemical Co.) at lgỈmL)1 in NaCl ⁄ Pi for Fluorescent nuclei were categorized according to the condensation and staining characteristics of chromatin Three random microscopic fields per sample of $ 100 nuclei were counted and mean values expressed as the percentage of apoptotic nuclei Caspase activity Caspase activity was determined in cytosolic protein extracts after harvesting and homogenization of cells in isolation buffer, containing 10 mm Tris-HCl buffer (pH 7.6), mm MgCl2, 1.5 mm potassium acetate, mm dithiothreitol and FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS P M Borralho et al protease inhibitor mixture tablets (Complete; Roche Applied Science, Mannheim, Germany) General caspase-3, -8 and -9-like activities were determined by enzymatic cleavage of chromophore p-nitroanilide (pNA) from the substrates N-acetyl-Asp-Glu-Val-Asp-pNA (DEVD-pNA), N-acetylIle-Glu-pro-Asp-pNA (IEPD-pNA) and N-acetyl-Leu-GluHis-Asp-p-NA (LEHD-pNA) (Sigma), respectively The proteolytic reaction was carried out in isolation buffer containing 50 lg of cytosolic protein and 50 lm specific caspase substrate The reaction mixtures were incubated at 37 °C for h, and the formation of pNA was measured by monitoring A405 using a 96-well plate reader miR-143 modulates 5-FU cytotoxicity Super SignalÔ substrate (Pierce, Rockford, IL, USA) b-actin (Sigma) was used as a loading control Protein concentrations were determined using the Bio-Rad protein assay kit according to the manufacturer’s instructions Statistical analysis All data are expressed as the mean ± SEM from at least three independent experiments Statistical significance was evaluated using the Student’s t-test P < 0.05 was considered statistically significant Acknowledgements miR-143 expression Total and miRNA-enriched RNA was extracted from cells using the mirVanaÔ PARIS kit from Ambion (Austin, TX, USA), recovering either small-RNA containing total RNA (total RNA) or small RNA species of less than 200 nucleotides (miRNA-enriched RNA) In addition, total RNA was also extracted using Trizol reagent (Invitrogen) RNA concentration was determined by monitoring A260 For real-time PCR, RT reactions were performed using a TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA assays for hsa-miR-143 and human RNU6B for normalization to endogenous control Data were collected with 7000 System Sequence Detection Software, version 1.2.3 (Applied Biosystems) Triplicate reactions were run per sample The comparative threshold cycle method was used to calculate the amplification factor where the threshold cycle (Ct) is defined as the cycle number at which the fluorescence passes the fixed threshold intensity level miR-143 expression levels in different samples were calculated on the basis of DDCt method HCT116 or SW480 parental untreated (control) cells were used as calibrator; the n-fold change in miR-143 expression was obtained using the formula: 2ÀDDCt Immunoblotting Steady-state levels of ERK5, NF-jB (p65) and Bcl-2 protein were determined by immunoblot analysis Briefly, 50 lg of total protein extracts were separated on 8% or 12% SDS-polyacrylamide electrophoresis gels After electrophoretic transfer onto nitrocellulose membranes, immunoblots were incubated with 15% H2O2 for 15 at room temperature After blocking with 5% milk solution, the blots were incubated overnight at °C with primary rabbit polyclonal antibody reactive to ERK5 (#3372; Cell Signaling, Beverly, MA, USA) and a mouse monoclonal antibody reactive to NF-jB (p65) or to Bcl-2 (#sc-8008 and #sc7382, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) Finally, membranes were incubated with secondary anti-mouse ⁄ -rabbit sera conjugated with horseradish peroxidase (Bio-Rad) for h at room temperature The membranes were processed for protein detection using The authors thank Dr Christine Esau, ISIS Pharmaceuticals Inc., for the kind gift of miR-143 overexpression and sensor plasmids The study was supported by grants FCG 68796 ⁄ 2004 from Fundacao Calouste ¸ ˜ Gulbenkian and PTDC ⁄ SAU-GMG ⁄ 099161 ⁄ 2008 from Fundacao para a Ciencia e a Tecnologia (FCT), ¸ ˜ ˆ Lisbon, Portugal (to C.M.P.R.); by PhD fellowship SFRH ⁄ BD ⁄ 24165 ⁄ 2005 (to P.M.B.) from FCT; and by postdoctoral fellowship SFRH ⁄ BPD ⁄ 30257 ⁄ 2006 (to R.E.C) from FCT References Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function Cell 116, 281–297 Carthew RW (2006) Gene regulation by microRNAs Curr Opin Genet Dev 16, 203–208 Michael MZ, SM OC, van Holst Pellekaan NG, Young GP & James RJ (2003) Reduced accumulation of specific microRNAs in colorectal neoplasia Mol Cancer Res 1, 882–891 Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M et al (2006) A microRNA expression signature of human solid tumors defines cancer gene targets Proc Natl Acad Sci USA 103, 2257–2261 Akao Y, Nakagawa Y & Naoe T (2006) MicroRNAs 143 and 145 are possible common onco-microRNAs in human cancers Oncol Rep 16, 845–850 Akao Y, Nakagawa Y, Kitade Y, Kinoshita T & Naoe T (2007) Downregulation of microRNAs-143 and -145 in B-cell malignancies Cancer Sci 98, 1914–1920 Lin T, Dong W, Huang J, Pan Q, Fan X, Zhang C & Huang L (2009) MicroRNA-143 as a tumor suppressor for bladder cancer J Urol 181, 1372–1380 Lui WO, Pourmand N, Patterson BK & Fire A (2007) Patterns of known and novel small RNAs in human cervical cancer Cancer Res 67, 6031–6043 Esau C, Kang X, Peralta E, Hanson E, Marcusson EG, Ravichandran LV, Sun Y, Koo S, Perera RJ, Jain R FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS 6699 miR-143 modulates 5-FU cytotoxicity 10 11 12 13 14 15 16 17 18 19 20 P M Borralho et al et al (2004) MicroRNA-143 regulates adipocyte differentiation J Biol Chem 279, 52361–52365 Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S & Miyazono K (2009) Modulation of microRNA processing by p53 Nature 460, 529–533 Chen X, Guo X, Zhang H, Xiang Y, Chen J, Yin Y, Cai X, Wang K, Wang G, Ba Y et al (2009) Role of miR-143 targeting KRAS in colorectal tumorigenesis Oncogene 28, 1385–1392 Akao Y, Nakagawa Y, Iio A & Naoe T (2009) Role of microRNA-143 in Fas-mediated apoptosis in human T-cell leukemia Jurkat cells Leuk Res 33, 1530–1538 Ng EK, Tsang WP, Ng SS, Jin HC, Yu J, Li JJ, Rocken C, Ebert MP, Kwok TT & Sung JJ (2009) MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer Br J Cancer 101, 699–706 Cordes KR, Sheehy NT, White MP, Berry EC, Morton SU, Muth AN, Lee TH, Miano JM, Ivey KN & Srivastava D (2009) miR-145 and miR-143 regulate smooth muscle cell fate and plasticity Nature 460, 705–710 Wang X & Tournier C (2006) Regulation of cellular functions by the ERK5 signalling pathway Cell Signal 18, 753–760 Pearson G, English JM, White MA & Cobb MH (2001) ERK5 and ERK2 cooperate to regulate NF-jB and cell transformation J Biol Chem 276, 7927–7931 Cude K, Wang Y, Choi HJ, Hsuan SL, Zhang H, Wang CY & Xia Z (2007) Regulation of the G2-M cell cycle progression by the ERK5-NFjB signaling pathway J Cell Biol 177, 253–264 Yu YY, Li Q & Zhu ZG (2005) NF-jB as a molecular target in adjuvant therapy of gastrointestinal carcinomas Eur J Surg Oncol 31, 386–392 Longley DB, Harkin DP & Johnston PG (2003) 5-fluorouracil: mechanisms of action and clinical strategies Nat Rev Cancer 3, 330–338 Borralho PM, Moreira da Silva IB, Aranha MM, Albuquerque C, Nobre Leitao C, Steer CJ & Rodrigues CM (2007) Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53 Biochim Biophys Acta 1772, 40–47 6700 21 Longley DB, Allen WL, McDermott U, Wilson TR, Latif T, Boyer J, Lynch M & Johnston PG (2004) The roles of thymidylate synthase and p53 in regulating Fas-mediated apoptosis in response to antimetabolites Clin Cancer Res 10, 3562–3571 22 Rossi L, Bonmassar E & Faraoni I (2007) Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro Pharmacol Res 56, 248–253 23 Petit E, Milano G, Levi F, Thyss A, Bailleul F & Schneider M (1988) Circadian rhythm-varying plasma concentration of 5-fluorouracil during a five-day continuous venous infusion at a constant rate in cancer patients Cancer Res 48, 1676–1679 24 Nakajima G, Hayashi K, Xi Y, Kudo K, Uchida K, Takasaki K, Yamamoto M & Ju J (2006) Non-coding microRNAs hsa-let-7g and hsa-miR-181b are associated with chemoresponse to S-1 in colon cancer Cancer Genomics Proteomics 3, 317–324 25 Takagi T, Iio A, Nakagawa Y, Naoe T, Tanigawa N & Akao Y (2009) Decreased expression of microRNA-143 and -145 in human gastric cancers Oncology 77, 12–21 26 Sachdeva M, Zhu S, Wu F, Wu H, Walia V, Kumar S, Elble R, Watabe K & Mo YY (2009) p53 represses c-Myc through induction of the tumor suppressor miR-145 Proc Natl Acad Sci USA 106, 3207–3212 27 Akao Y, Nakagawa Y & Naoe T (2007) MicroRNA143 and -145 in colon cancer DNA Cell Biol 26, 311– 320 28 Nishimoto S & Nishida E (2006) MAPK signalling: ERK5 versus ERK1 ⁄ EMBO Rep 7, 782–786 29 Pi X, Yan C & Berk BC (2004) Big mitogen-activated protein kinase (BMK1) ⁄ ERK5 protects endothelial cells from apoptosis Circ Res 94, 362–369 30 Girio A, Montero JC, Pandiella A & Chatterjee S (2007) Erk5 is activated and acts as a survival factor in mitosis Cell Signal 19, 1964–1972 31 Nakagawa Y, Iinuma M, Naoe T, Nozawa Y & Akao Y (2007) Characterized mechanism of alpha-mangostininduced cell death: caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression in human colorectal cancer DLD-1 cells Bioorg Med Chem 15, 5620–5628 FEBS Journal 276 (2009) 6689–6700 ª 2009 The Authors Journal compilation ª 2009 FEBS ... results obtained indicated that mature miR-143 enhanced sensitivity to 5-FU Indeed, cell viability was reduced and cell death was increased in HCT116- OV3 compared to parental and HCT116- EM1 cells, ... growth and ⁄ or escape from apoptosis In addition, miR-143 expression has been shown to increase after a-mangostin exposure in human colon cancer DLD-1 cells, resulting in increased apoptosis... evaluated in situ in mouse colonic epithelial crypts [27] and found mainly in the cytoplasm, where it was more intense in the ascending more differentiated pre-apoptotic glandular cells than in the