Dottorato in BIOCHIMICA

Titolo della tesi: Impact of β-hexachlorocyclohexane on human cellular biochemistry and environmental remediation strategies.

Environmental pollution represents one of the most pressing problems in developed countries and in recent years has raised concern and doubts also from the scientific perspective. In fact, an ever-growing number of epidemiologic-observational studies, carried out on population at risk, correlated the exposure to environmental chemicals with the incidence of several pathological conditions, ranging from metabolic to cardiological and reproductive diseases, until the development of cancers. These evidences have made more urgent the need for further investigations on the biological mechanism at the basis of pollutants toxicity. In particular, significant attention has been paid to evaluating the impact of organochlorine pesticides (OCPs) on human health. OCPs belong to a large class of organic compounds that the Stockholm Convention catalogued as “POPs” (Persistent Organic Pollutants). The list of banned chemicals includes dioxins and their derivatives, hexachlorocyclohexane, polychlorinated biphenyls and aldrin, whereas many other similar substances are subjected to restrictions. OCPs are widely distributed in the biosphere and their hazardousness is mostly related to physicochemical properties such as lipophilia and energetic stability, that allow these molecules to be resistant to biodegradation and to bio-accumulate into the adipose tissue. Information about the molecular mechanisms of the most popular OCPs (i.e. dioxins, DDT) is already present in scientific literature and several studies indicated them as endocrine disrupting chemicals as well as oncogenes. On the other hand, not much is known about a dangerous and widely diffused compound: the hexachlorocyclohexane. Hexachlorocyclohexane (HCH) is a chlorinated cyclic saturated hydrocarbon that exists in four isomers: α, β, γ and δ hexachlorocyclohexane. The g-isomer of HCH, also known as lindane, is a broad-spectrum insecticide that has been extensively used for the control of agricultural pests and for health purposes. Among HCH isomers, which are by-products of lindane industrial synthesis, β-HCH is the most recalcitrant because of its higher energetic stability due to the equatorial position of all six chlorine atoms in the chair cyclohexane conformation; in addition, few reports are available about its metabolic breakdown. For this characteristic, it is usually the predominant isomer remaining in soils and in animal tissue and can still be detected at low background environmental levels. The improper disposal of huge amounts of β-HCH led to the generation of contaminated sites in several parts of the world (Italy, Turkey, Spain, Kazakhstan, Canada, India, China, Russia, Poland, Germany, Argentine): this classifies “lindane’s contamination” as one of the environmental catastrophe of global proportions on the planetary scale. A detailed epidemiological study, ongoing since 2006, has found correlation between high blood levels of β-HCH and the occurrence of a wide range of diseases in a sample of 660 exposed patients living close the Valle del Sacco, south of Rome. The Valle del Sacco, in fact, is characterized by the presence of a large industrial conglomerate in which lindane production has been stopped in 70’s. Although the biomonitoring study highlighted a link between β-HCH contamination and the incidence of several pathological conditions, few data are currently available in the scientific literature regarding the molecular mechanism of β-HCH. For this reason, our laboratory is investigating since 2015 the intracellular effects of β-HCH with a particular focus on its impact on cancer cells. In a first published study, experiments were carried out on a panel of cells representing different human tumor types (i.e. liver, lungs, prostate, breast) associated with the expression and activation of specific receptors or kinases that are related to STAT3 activity. The experimental concentration of 10 µM for β-HCH was chosen averaging across all the plasma concentration values detected in patients under the biomonitoring study carried out in the Valle del Sacco, in order to reproduce the real exposure conditions. After evaluating the effects of β-HCH on cellular viability, different types of analysis were performed to identify the transduction cascades involved in the molecular responses to β-HCH. Obtained results established that β-HCH can activate cell-line specific pathways that all converge in STAT3 activation. Then, a special focus was placed on investigating the putative role of β-HCH in prostate cancer progression; in fact, literature data, together with our previous findings, suggest that β-HCH could have an endocrine disrupting activity by interfering with Androgen Receptor (AR) signaling. To confirm this hypothesis, LNCaP cells (hormone-sensitive prostate cancer cell line) were treated with β-HCH or testosterone in the presence or absence of the chemotherapeutic agent bicalutamide. The outcomes show that AR nuclear translocation occurs upon both β-HCH and testosterone treatment, whereas is inhibited in the presence of bicalutamide, as evidenced by immunoblotting analysis on nuclear extracts and immunofluorescence experiments. Subsequently, was verified whether β-HCH could affect the activity of AhR (Aryl Hydrocarbon Receptor), the xenobiotic sensor par excellence, in both hormone-dependent and independent tumor types. Immunofluorescence analysis evidenced the capability of β-HCH to induce AhR nuclear translocation. In addition, immunoblotting analysis were performed on cells treated with β-HCH in the presence or not of MG-132 (proteasome inhibitor) and CH223191 (AhR inhibitor) and obtained results clearly highlighted the influence of β-HCH on AhR signaling. Then, experiments were performed to investigate whether β-HCH, on par with other organochlorine pesticides, can induce oxidative stress. For this purpose, ROS production and GSSG/GSH ratio were measured, evidencing the impact of β-HCH on cellular redox homeostasis. In parallel, variations in cellular bioenergetic profile were monitored, demonstrating that β-HCH promote a metabolic shift toward aerobic glycolysis. In this altered context, β-HCH can also induce DNA damage through H2AX phosphorylation. Subsequently, the potential role of β-HCH as a contributor in tumor initiation was inspected. Experiments were carried out on a continuous normal bronchial epithelium cell line to investigate whether β-HCH could trigger cellular malignant transformation toward cancer development. For this reason, β-HCH impact was evaluated on cells viability and morphology and some markers for tumorigenesis, as Ki67 positive-cells and EGF secretion, were studied along with β-HCH activation pathways. Experimental outcomes strongly support the oncogenic potential of this molecule. Considering the capability of β-HCH to promote cell growth and tumor progression, the next question to answer is whether the exposure to β-HCH may lead to a loss of response to chemotherapeutic agents such as tyrosine kinases inhibitors. Experiments carried out on a HER2-positive lung cancer cell line revealed that β-HCH can counteract the inhibitory activity of lapatinib, leading to a higher cell proliferation rate via STAT3 activation. Further investigations were conducted using other chemotherapeutic agents (cisplatin, camptothecin and paclitaxel) and preliminar results seem to confirm the loss of sensitivity to drugs in the presence of β-HCH. From an environmental point of view, the persistence of β-HCH still represents an open question for the presence of massive illegal repositories all around the world. For this reason, β-HCH degradation through a copper-based Fenton-like method was explored by setting up a HPLC protocol under different experimental conditions. The process focused on the quantitative degradation of the parental β-HCH, since the detection of its breakdown products or transformed molecules would need a mass-spectrometry for their qualitative characterization. In parallel with the β-HCH research topic, the role of the protein STAT3 in prostate cancer was further deepened. STAT3 (Signal Transducer and Activator of Transcription 3) is a converging point for many signaling cascades and has been reported constitutively activated in a wide range of solid tumors and hematological malignancies. STAT3 is a latent cytosolic transcription factor and upregulates the expression of genes involved in cell survival and proliferation upon a wide variety of stimuli, including cytokines, oncogenes, growth factors or cytosolic kinases. The dynamic biological behavior of STAT3 can explain the higher proliferation rate triggered by β-HCH through the activation of STAT3-mediated pathways. STAT3 fulfils its multifaceted molecular functions through two different intracellular mechanisms, generally referred as canonical and non-canonical pathways. The canonical activation of STAT3 is strictly dependent on its phosphorylation at the tyrosine residue 705; upon phosphorylation at Y705, induced by the binding of a ligand to its receptor, STAT3 undergoes homodimerization to form an active dimer that can translocate to nucleus and mediates its transcriptional activity. Besides its well-described canonical signaling, STAT3 can be subjected to alternative post-translational modifications. In addition, recent studies assessed the involvement of STAT3, by means of both its canonical and non-canonical pathway, in the metabolic shift toward aerobic glycolysis known as Warburg Effect, which is typical of the more aggressive tumor phenotypes. On the basis of these premises, the existence of a link between PTMs and specific STAT3-mediated pathways was investigated in LNCaP (less aggressive PCa form) and DU-145 (more aggressive) cells performing experiments that simulated inflammatory and oxidative-stress conditions. Cells were treated with IL-6 to induce an inflammatory response, whereas tert-butyl hydroperoxide (t-BHP) was used to simulate oxidative stress. Obtained results on cellular models confirmed the relationship between STAT3 PTMs and cellular conditions, thereby reinforcing the hypothesis that PTMs can drive intracellular responses through STAT3-mediated signaling pathways. Thus, it is possible to identify STAT3 PTMs and STAT3 modulators as suitable markers or targets for PCa prevention, diagnosis and therapy. Then the role of STAT3 in prostate cancer energy metabolism was further investigated, with particular focus on the protein SHMT2 (Serine-Hydroxymethyltransferase). Results indicate that SHMT2 is an active player in STAT3 signaling and that its expression is upregulated by the JAK2/STAT3 canonical pathway upon IL-6 stimulation. Experiments were carried out on two different prostate cancer cell lines, LNCaP (less aggressive) and DU145 (more aggressive). The observation was extended to PCa formalin-fixed paraffin-embedded (FFPE) tissue sections obtained from total prostatectomies: collected specimens are characterized by a different Gleason score, ranging from 6 (less aggressive) to 9 (more aggressive). In both cell lines, STAT3 activation mode, the amount and distribution of PKM2, SHMT2, and HIF-1a proteins, as well as the cellular metabolic conditions, were evaluated in the presence or absence of IL-6-induced inflammation. Expression levels of PKM2, SHMT2, and HIF-1a, together with interleukin-6, were also analyzed utilizing normal and tumor FFPE tissues.