Raquel T. Lima

MicroRNA regulation of core apoptosis pathways in cancer

Recent research has demonstrated that microRNAs (miRNAs) are key regulators of many cell processes often deregulated in cancer, including apoptosis. Indeed, it is becoming clear that many miRNAs are anti-apoptotic and mediate this effect by targeting pro-apoptotic mRNAs or positive regulators of pro-apoptotic mRNAs. Conversely, many pro-apoptotic miRNAs target anti-apoptotic mRNAs or their positive regulators. We have reviewed the current knowledge in this area including evidence of miRNA involvement in cancer drug resistance.

Anti-hepatocellular carcinoma activity using human HepG2 cells and hepatotoxicity of 6-substituted methyl 3-aminothieno[3,2-b]pyridine-2- carboxylate derivatives: In vitro evaluation, cell cycle analysis and QSAR studies

Hepatocellular carcinoma (HCC) is a highly complex cancer, resistant to commonly used treatments and new therapeutic agents are urgently needed. A total of thirty-two thieno[3,2-b]pyridine derivatives of two series: methyl 3-amino-6-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates (1a-1t) and methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]pyridine-2-carboxylates (2a-2n), previously prepared by some of us, were evaluated as new potential anti-HCC agents by studying their in vitro cell growth inhibition on human HepG2 cells and hepatotoxicity using a porcine liver primary cell culture (PLP1). The presence of amino groups linked to a benzene moiety emerges as the key element for the anti-HCC activity. The methyl 3-amino-6-[(3-aminophenyl)ethynyl]thieno[3,2-b]pyridine-2-carboxylate (2f) is the most potent compound presenting GI(50) values on HepG2 cells of 1.2 μM compared to 2.9 μM of the positive control ellipticine, with no observed hepatotoxicity (PLP1 GI(50) > 125 μM against 3.3 μM of ellipticine). Moreover this compound changes the cell cycle profile of the HepG2 cells, causing a decrease in the % of cells in the S phase and a cell cycle arrest in the G2/M phase. QSAR studies were also performed and the correlations obtained using molecular and 1D descriptors revealed the importance of the presence of amino groups and hydrogen bond donors for anti-HCC activity, and hydrogen bond acceptors for hepatotoxicity. The best correlations were obtained with 3D descriptors belonging to different subcategories for anti-HCC activity and hepatotoxicity, respectively. These results point to different molecular mechanisms of action of the compounds in anti-HCC activity and hepatotoxicity. This work presents some promising thieno[3,2-b]pyridine derivatives for potential use in the therapy of HCC. These compounds can also be used as scaffolds for further synthesis of more potent analogs.

miR-143 Overexpression Impairs Growth of Human Colon Carcinoma Xenografts in Mice with Induction of Apoptosis and Inhibition of Proliferation

Background MicroRNAs (miRNAs) are aberrantly expressed in human cancer and involved in the (dys)regulation of cell survival, proliferation, differentiation and death. Specifically, miRNA-143 (miR-143) is down-regulated in human colon cancer. In the present study, we evaluated the role of miR-143 overexpression on the growth of human colon carcinoma cells xenografted in nude mice (immunodeficient mouse strain: N: NIH(s) II-nu/nu). Methodology/Principal Findings HCT116 cells with stable miR-143 overexpression (Over-143) and control (Empty) cells were subcutaneously injected into the flanks of nude mice, and tumor growth was evaluated over time. Tumors arose ∼ 14 days after tumor cell implantation, and the experiment was ended at 40 days after implantation. miR-143 was confirmed to be significantly overexpressed in Over-143 versus Empty xenografts, by TaqMan® Real-time PCR (p<0.05). Importantly, Over-143 xenografts displayed slower tumor growth compared to Empty xenografts from 23 until 40 days in vivo (p<0.05), with final volumes of 928±338 and 2512±387 mm3, respectively. Evaluation of apoptotic proteins showed that Over-143 versus Empty xenografts displayed reduced Bcl-2 levels, and increased caspase-3 activation and PARP cleavage (p<0.05). In addition, the incidence of apoptotic tumor cells, assessed by TUNEL, was increased in Over-143 versus Empty xenografts (p<0.01). Finally, Over-143 versus Empty xenografts displayed significantly reduced NF-κB activation and ERK5 levels and activation (p<0.05), as well as reduced proliferative index, evaluated by Ki-67 immunohistochemistry (p<0.01). Conclusions Our results suggest that reduced tumor volume in Over-143 versus Empty xenografts may result from increased apoptosis and decreased proliferation induced by miR-143. This reinforces the relevance of miR-143 in colon cancer, indicating an important role in the control of in vivo tumor progression, and suggesting that miR-143 may constitute a putative novel therapeutic tool for colon cancer treatment that warrants further investigation.

Targeting miR-21 Induces Autophagy and Chemosensitivity of Leukemia Cells

Overexpression of oncomiR-21 has been observed in most cancer types, such as leukemia. This miR has been implicated in a number of cellular processes, including chemoresistance, possibly by directly modulating the expression of several apoptotic related proteins. It was recently shown to directly target Bcl-2 mRNA and upregulate Bcl-2 protein expression. Nevertheless, the possible effect of miR-21 in autophagy has never been addressed. This study investigates the effects of targeting miR-21 with antimiRs on chronic myeloid leukemia cellular autophagy and on associated drug sensitivity. We observed that miR-21 downregulation decreased cellular viability and proliferation, although no changes to the normal cell cycle profile were observed. miR-21 downregulation also caused increased programmed cell death and a decrease in the expression levels of Bcl-2 protein, although PARP cleavage was not affected, indicating that apoptosis was not the relevant mechanism underlying the observed results. Treatment with antimiR-21 caused an increase in the autophagy related proteins Beclin-1, Vps34 and LC3-II. Accordingly, autophagic vacuoles were visualized both by monodansylcadaverine (MDC) and acridine orange (AO) staining and also by transmission electron microscopy (TEM). Additionally, miR-21 downregulation increased K562 and KYO-1 cellular sensitivity to etoposide or doxorubicin. This chemosensitivity was reverted by pre-treating cells with 3-MA, an autophagy inhibitor. Finally, serum starvation (an autophagy inducer) also increased sensitivity to these drugs, confirming that autophagy sensitized these cells to the effect of these drugs. To the best of our knowledge, this is the first description of autophagy induction via miR-21 targeting and its involvement in drug sensitivity.

Prenylated derivatives of baicalein and 3,7-dihydroxyflavone: Synthesis and study of their effects on tumor cell lines growth, cell cycle and apoptosis

Fourteen baicalein and 3,7-dihydroxyflavone derivatives were synthesized and evaluated for their inhibitory activity against the in vitro growth of three human tumor cell lines. The synthetic approaches were based on the reaction with prenyl or geranyl bromide in alkaline medium, followed by cyclization of the respective monoprenylated derivative. Dihydropyranoflavonoids were also obtained by one-pot synthesis, using Montmorillonite K10 clay as catalyst combined with microwave irradiation. In vitro screening of the compounds for cell growth inhibitory activity revealed that the presence of one geranyl group was associated with a remarkable increase in the inhibitory activity. Moreover, for the 3,7-dihydroxyflavone derivatives a marked increase in growth inhibitory effect was also observed for compounds with furan and pyran fused rings. The most active compounds were also studied regarding their effect on cell cycle profile and induction of apoptosis. Overall the results point to the relevant role of the prenylation of flavone scaffold in the growth inhibitory activity of cancer cells.

Insights into the In Vitro Antitumor Mechanism of Action of a New Pyranoxanthone

Naturally occurring xanthones have been documented as having antitumor properties, with some of them presently undergoing clinical trials. In an attempt to improve the biological activities of dihydroxyxanthones, prenylation and other molecular modifications were performed. All the compounds reduced viable cell number in a leukemia cell line K‐562, with the fused xanthone 3,4‐dihydro‐12‐hydroxy‐2,2‐dimethyl‐2H,6H‐pyrano[3,2‐b]xanthen‐6‐one (5) being the most potent. The pyranoxanthone 5 was particularly effective in additional leukemia cell lines (HL‐60 and BV‐173). Furthermore, the pyranoxanthone 5 decreased cellular proliferation and induced an S‐phase cell cycle arrest. In vitro, the pyranoxanthone 5 increased the percentage of apoptotic cells which was confirmed by an appropriate response at the protein level (e.g., PARP cleavage). Using a computer screening strategy based on the structure of several anti‐ and pro‐apoptotic proteins, it was verified that the pyranoxanthone 5 may block the binding of anti‐apoptotic Bcl‐xL to pro‐apoptotic Bad and Bim. The structure‐based screening revealed the pyranoxanthone 5 as a new scaffold that may guide the design of small molecules with better affinity profile for Bcl‐xL.

The in vitro anticancer activity of the crude extract of the sponge-associated fungus Eurotium cristatum and its secondary metabolites

Background: Marine natural products has captivated many researchers over the years and there is always a need for sources of diverse and pharmacologically active leads in the area of anticancer drugs. Materials and Methods: The ethyl acetate extract of the fungus Eurotium cristatum (ECE), isolated from the marine sponge Mycale sp., furnished 2-(2’, 3-epoxy-1’,3’-heptadienyl)-6-hydroxy-5-(3-methyl-2-butenyl) benzaldehyde (1), 1,8-dihydroxy-6-methoxy-3-methyl-9,10-anthracenedione (physcion, 2), and the dioxopiperazine alkaloid echinulin (3). The structures of the compounds were established by Nuclear Magnetic Resonance (NMR) spectral analysis (1H, 13C, DEPT, COSY, HSQC, and HMBC). The ECE and its metabolites were evaluated for their growth inhibitory activity on the following three human tumor cell lines: breast adenocarcinoma (MCF-7), non-small lung cancer (NCI-H460), and melanoma (A375-C5). Results: The results showed that the ECE was active in all the three cell lines, with the values of GI50 = 44.3 ± 1.2, 45.5 ± 7.5, and 71.3 ± 2.1 μg/ml for MCF-7, NCI-H460, and A375-C5, respectively. Compound 1 also exhibited moderate growth inhibitory activity against all the three cell lines (GI50 = 58.3 ± 1.2, 46.0 ± 5.5, and 116.7 ± 7.2 μM for MCF-7, NCI-H460, and A375-C5, respectively), whereas compound 3 showed only weak inhibition against MCF-7 (GI50 = 109.7 ± 0.3 μM) and NCI-H460 (GI50 = 96.7 ± 1.5 μM) but was inactive against A375-C5 (GI50 >150 μM). On the contrary, compound 2 was inactive in all the three cell lines at the highest concentration tested (150 μM). Furthermore, ECE was investigated for its effect on the cell cycle in the NCI-H460 cells. Analysis of the cell cycle profile showed that ECE was able to cause a slight cell arrest in the G1 phase, with a corresponding decrease of cells in the S and G2/M phases. Conclusion: The secondary metabolites isolated [Compound 1] from the crude ethyl acetate extract of the culture of the marine fungus E. cristatum were found as the most potent compound regarding cell growth inhibition.

The network of P-glycoprotein and microRNAs interactions.

Overexpression of P‐glycoprotein (P‐gp) contributes to the multidrug resistance (MDR) phenotype found in many cancer cells. P‐gp has been identified as a promising molecular target, although attempts to find successful therapies to counteract its function as a drug efflux pump have largely failed to date. Apart from its role in drug efflux, P‐gp may have other cellular functions such as being involved in apoptosis, and is found in various locations in the cell. Its expression is highly regulated, namely by microRNAs (miRNAs or miRs). In addition, P‐gp may regulate the expression of miRs in the cell. Furthermore, both P‐gp and miRs may be found in microvesicles or exosomes and may be transported to neighboring, drug‐sensitive cells. Here, we review this current issue together with recent evidence of this network of interactions between P‐gp and miRs.

Efficient synthesis of 6-(hetero)arylthieno[3,2-b]pyridines by Suzuki-Miyaura coupling. Evaluation of growth inhibition on human tumor cell lines, SARs and effects on the cell cycle.

A wide variety of new bi(hetero)aryl derivatives of the thieno[3,2-b]pyridine skeleton was obtained in high to excellent yields (65-91%) by Suzuki-Miyaura cross-coupling of the methyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate, recently reported by us, with aryl or heteroaryl pinacolboranes or potassium trifluoroborates. The coupling products obtained were evaluated for their growth inhibitory effect on three human tumor cell lines, representing different tumor models, MCF-7 (breast adenocarcinoma), A375-C5 (melanoma) and NCI-H460 (non-small cell lung cancer). Some of the compounds showed an interesting activity against the tested cell lines, with GI50 values in the μM range, and it was possible to establish some structure-activity relationships (SARs). Several compounds presented GI50 values below 15 μM, particularly a bithiophene and an o-aniline thienopyridine derivative. The first presented selectivity for MCF-7 and NCI-H460 cell lines, with very low GI50 values (0.7-1.0 μM), while the latter was active against the three cell lines tested in this study, also presenting very low GI50 values (2.5-4.2 μM). The effect of these two compounds on cell cycle progression was analyzed in the NCI-H460 cell line. Results showed that both compounds interfered with the normal cell cycle distribution.

Intercellular Transfer of Cancer Drug Resistance Traits by Extracellular Vesicles

Extracellular vesicles (EVs) are nanosized particles (100-1000 nm) enclosed by a phospholipid bilayer that have been described as important mediators of intercellular communication. The role of EVs in oncobiology has been extensively studied, including their contribution to the horizontal transfer of drug resistance from drug-resistant to drug-sensitive cancer cells. This review focuses on the EVs cargo responsible for this intercellular transfer of drug resistance; namely, drug-efflux pumps, miRNAs, long noncoding RNAs (lncRNAs), and other mediators. Additionally, the known molecular mechanisms and features of this transfer are discussed. This is an emerging area of research and we highlight topics that need to be further studied to fully understand and counteract the intercellular transfer of drug resistance mediated by EVs.