Juan Sebastian Yakisich

Natural products targeting autophagy via the PI3K/Akt/mTOR pathway as anticancer agents.

The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is a key regulator of authophagy. Natural products show anticancer activity and often induce apoptosis or autophagy. The crosstalk between these two types of cell death makes autophagy an interesting target since drugs targeting this process not only can induce cell death by inducing autophagy but can also sensitize cells to apoptosis. Autophagy is also a protective mechanism associated with increased resistance to chemotherapy. In this review, we discuss natural products known to induce autophagy cell death in cancer cells via the PI3K/Akt/mTOR pathway.

Role of apoptosis-related miRNAs in resveratrol-induced breast cancer cell death.

Breast cancer is the most frequently diagnosed cancer in women, and one of the leading causes of cancer-related deaths worldwide. Recent evidences indicate that dietary agents such as resveratrol may inhibit cancer progression through modulation of microRNAs (miRNAs). We demonstrate that resveratrol regulates apoptotic and cell cycle machinery in breast cancer cells by modulating key tumor-suppressive miRNAs including miR-125b-5p, miR-200c-3p, miR-409-3p, miR-122-5p and miR-542-3p. Resveratrol-mediated miRNA modulation regulates key anti-apoptotic and cell cycle proteins including Bcl-2, X-linked inhibitor of apoptosis protein and CDKs, which are critical for its activity. Modulating miRNAs with mimics or inhibitors further validated a key role for miR-542-3p in MCF-7 and miR-122-5p in MDA-MB-231 breast cancer cell death in response to resveratrol. In conclusion, this study reveals novel miRNAs modulated by resveratrol that have a key role in breast cancer cell death.

Inhibition of DNA synthesis in human gliomas by roscovitine

The early effect of 1-100 microM roscovitine, a purine analogue and cyclin-dependent kinase inhibitor, was studied on tissue specimens from eight human malignant gliomas. The tissue was incubated immediately after resection with DMEM containing [3H]methylthymidine plus vehicle alone or the proper concentration of roscovitine for 30-90 min. The DNA synthesis rate was assessed by measurement of [3H]methylthymidine incorporation into trichloroacetic acid insoluble material/mg protein/min. In all gliomas, 100 microM roscovitine inhibited DNA synthesis by 71-97% (average 89 +/- 8%, p<0.0001). This inhibitory effect of roscovitine appeared within 30 min of incubation and was concentration dependent.

The Stemness Phenotype Model

The identification of a fraction of cancer stem cells (CSCs) associated with resistance to chemotherapy in most solid tumors leads to the dogma that eliminating this fraction will cure cancer. Experimental data has challenged this simplistic and optimistic model. Opposite to the classical cancer stem cell model, we introduced the stemness phenotype model (SPM), which proposed that all glioma cells possess stem cell properties and that the stemness is modulated by the microenvironment. A key prediction of the SPM is that to cure gliomas all gliomas cells (CSCs and non-CSCs) should be eliminated at once. Other theories closely resembling the SPM and its predictions have recently been proposed, suggesting that the SPM may be a useful model for other type of tumors. Here, we review data from other tumors that strongly support the concepts of the SPM applied to gliomas. We include data related to: (1) the presence of a rare but constant fraction of CSCs in established cancer cell lines, (2) the clonal origin of cancer, (3) the symmetrical division, (4) the ability of “non-CSCs” to generate “CSCs,” and (5) the effect of the microenvironment on cancer stemness. The aforenamed issues that decisively supported the SPM proposed for gliomas can also be applied to breast, lung, prostate cancer, and melanoma and perhaps other tumors in general. If the glioma SPM is correct and can be extrapolated to other types of cancer, it will have profound implications in the development of novel modalities for cancer treatment.

The effect of phosphoinositide 3-kinase inhibitors on programmed nuclear degradation in Tetrahymena and fate of surviving nuclei

The effect of phosphoinositide 3-kinase inhibitors on programmed nuclear degradation in Tetrahymena and fate of surviving nuclei

Low doses of urban air particles from Buenos Aires promote oxidative stress and apoptosis in mice lungs

Air pollution consists of a wide range of gaseous and particulate pollutants. Exposure to particulate matter (PM) can cause oxidative stress within the lung, which in turn can negatively impact health. The mechanisms by which PM causes oxidative stress include the release of trace metals or organic components from the particle. Previously, we have characterized urban air particles from downtown Buenos Aires (UAP-BA) and, by using in vivo animal studies, found that they are able to generate lung inflammation. Purpose: We studied lung responses to low doses of UAP-BA (15 µg), with special emphasis on oxidative balance. Methods: We assessed cell viability, total cell number (TCN) and cell differential (CD) on bronchoalveolar lavages (BAL), oxidative metabolism in lung homogenates by tertbutylhydroperoxide-initiated chemiluminescence (CL), thiobarbituric reactive substances (TBARS), total reactive antioxidant potential (TRAP), reduced glutathione (GSH), and apoptosis in lung sections. Results: We found that low UAP-BA exposure increases TCN, modifies CD, and decreases cell viability in the BAL. In lung homogenates, TBARS and CL rose while TRAP and GSH showed no alteration when compared to controls. Occurrence of apoptosis evaluated by TUNEL assay was markedly augmented in UAP-BA exposed animals. Conclusions: Our data further implicate oxidative stress as a possible inducer of apoptosis in lungs from animals exposed to low concentrations of this urban environmental contaminant.

Simvastatin pretreatment prevents ambient particle-induced lung injury in mice

Air particulate pollution negatively affects the health of the population exposed, being the lung the main target organ. Simvastatin (SV) is widely used for the prevention and risk reduction of coronary disease. Its pleiotropic effects may provide benefit for lung diseases. Here, we investigated the preventive effect of simvastatin pretreatment on acute intranasal exposure to ROFA (Residual Oil Fly Ash), and UAP (Urban Air Particle from Buenos Aires). Male BALB/c mice were randomized in two groups to receive either saline (control, C) solution or SV (1 mg/kg bw /day; ip) for 14 days. After SV treatment, ROFA or UAP (1 mg/kg bw) or saline were intranasally delivered for 24 hours generating 4 subgroups for the ROFA experiment (C, SV, ROFA and SV+ROFA) and 3 subgroups for the UAP experiment (C, SV, UAP and SV+UAP). Biomarkers of lung injury were examined in BAL cells evaluating total cell number (TCN), cell differential (CD) and superoxide anion generation (O2-), in lung homogenates assessing superoxide dismutase activity (SOD) and tumor necrosis factor α (TNFα); and in blood samples determining interleukin 6 (IL-6) production. ROFA and UAP produced an acute pulmonary injury, characterized by an increase in BAL, TCN and neutrophilic inflammatory influx, a rise in O2- generation, and production of the proinflammatory TNFα cytokine. SV pretreatment had no significant effect per se on any of these biomarkers but prevented the pulmonary cytotoxicity and inflammation induced by ROFA and UAP. Our results encourage further studies to determine the preventive effects on lung injury induced by air pollutants.

A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label‐free LC‐MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO‐P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal‐regulated kinase (ERK) were identified to be key mediators of pro‐ and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO‐P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.

Anti-Tumor Effects of Cardiac Glycosides on Human Lung Cancer Cells and Lung Tumorspheres: ANTI-TUMOR EFFECTS OF CARDIAC GLYCOSIDES

Lung cancer is a leading cause of cancer‐related death in the United States. Although several drugs have been developed that target individual biomarkers, their success has been limited due to intrinsic or acquired resistance for the specific targets of such drugs. A more effective approach is to target multiple pathways that dictate cancer progression. Cardiac glycosides demonstrate such multimodal effects on cancer cell survival, and our aim was to evaluate the effect of two naturally occurring monosaccaridic cardiac glycosides—Convallatoxin and Peruvoside on lung cancer cells. Although both drugs had significant anti‐proliferative effects on H460 and Calu‐3 lung cancer cells, Convallatoxin demonstrated twofold higher activity as compared to Peruvoside using both viability and colony forming assays, suggesting a role for the aglycone region in dictating drug potency. The tumor suppressor p53 was found to be important for action of both drugs—p53‐underexpressing cells were less sensitive as compared to p53‐positive H460 cells. Further, assessment of p53‐underexpressing H460 cells showed that drugs were able to arrest cells in the G0/G1 phase of the cell cycle in a dose‐dependent manner. Both drugs significantly inhibited migration and invasion of cancer cells and decreased the viability of floating tumorspheres. An assessment of intracellular pathways indicated that both drugs were able to modulate proteins that are involved in apoptosis, autophagy, cell cycle, proliferation, and EMT. Our data suggest, a promising role for cardiac glycosides in lung cancer treatment, and provides impetus for further investigation of the anti‐cancer potential of this class of drugs. J. Cell. Physiol. 232: 2497–2507, 2017.

Formation of Tumorspheres with Increased Stemness without External Mitogens in a Lung Cancer Model

Like with most solid tumors, the presence of a subpopulation of cancer stem cells (CSCs) or cancer stem-like cells (CS-LCs) has been associated with chemoresistance and tumor relapse in lung cancer cells. In the absence of serum, CSCs/CS-LCs have the ability to grow as lung tumorspheres (LTSs), and this system is routinely used for isolation and characterization of putative CSCs/CS-LCs. Methods to isolate LTSs are usually performed in serum-free media supplemented with specific additives such as epidermal growth factor and basic fibroblast growth factor. In this study, we report the generation of LTSs without the addition of any external mitogenic stimulation. LTSs generated in this manner demonstrated several traits usually associated with increased stemness such as elevated expression of the stemness-associated marker Sox2 and increased chemoresistance to conventional anticancer drugs. In addition, we report that the FDA-approved drug Digitoxin, at concentration close to its therapeutic level, decreased the viability of LTSs and downregulated Sox2 independent of the PI3K/AKT pathway. The potential use of LTSs generated without the addition of any external mitogenic stimulation to study the role of specific factor(s) associated with stemness properties is also discussed.