Biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in PC3 cell line
Creators: name: Granato, Giovanna Elvira
Publisher: name: Università degli Studi di Napoli Federico II
Year: 2013

description: Dioxins are commonly known as highly toxic compounds that are persistent organic pollutants. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent dioxin congener,  and dioxin-like products are formed during the incomplete combustion of organic compounds in the presence of chlorine (waste incineration, burning of various fuels and poorly controlled combustion sources). In 1997, the International Agency for Research on Cancer (IARC) classified TCDD as carcinogen. 
Prostate cancer (PC) is an extremely serious disease in dogs. PC also represents the most commonly diagnosed cancer in males in the Western world. In general, animal models of human cancer have evolved in attempts to capture the complexity of the human disease. In particular, as humans, the dogs are the only other mammals that develop PC. The canine prostate gland shares many morphological and functional similarities with the human prostate, so this specie represents thus an attractive model for the study of the prostatic disease in dogs. Consequently, to evaluate the biological effects of TCDD on prostate cancer, in this study we used human prostate cancer cell line, PC3.  Until now, very few data are present in literature about PC3 and TCDD exposure. It is known that in PC3 cell line, a hormone-independent prostate cancer cell line, TCDD induces cytochrome P450 (CYP) 1A1 and CYP1B1 via the aryl hydrocarbon receptor (AhR). This effect of TCDD could result in higher elimination rates of concomitant drugs metabolized by these particular CYP isoenzymes.
Autophagy is a tightly regulated process playing a normal part in cell growth, development, and homeostasis and helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products through the degradation via the lysosome. Defects of autophagy machinery are responsible for pathogenesis of different diseases, including cancer. The role of autophagy in cancer is controversial. There is evidence that autophagy may play a critical role in cancer progression at later stages, such as dissemination and metastasis, which account for most cancer-associated deaths, whereas in other cases it clearly contributes to tumor suppression by inducing tumor cell death. So, the aim of this study is to evaluate the biological effects of TCDD exposure to a highly metastatic prostate cancer cell line, PC3. 
 The prostate cancer cell line PC3 was exposed to different concentrations of TCDD (0.1, 1 and 100 pg/ml) and after 24, 48, and 72 hours of exposure cell proliferation and  viability, cell morphology and cell cycle analysis were performed. Also, autophagy was evaluated using the following: (a) detection of acidic vesicular organelles (AVOs) by acridine orange staining; (b) immunofluorescence analysis (IF) of LC3 (microtubule associated light chain protein 3), one of the well-known autophagy markers; (c) LC3 gene expression by real-time PCR; (d) study of autophagic flux by chloroquine (CQ) (10µM), autophagy inhibitor, by western blot; (e) study the expression of multiple genes involved in autophagy machinery by real-time PCR.
	 When compared with their relative controls, TCDD (100 pg/ml) exposure at 24, 48, and 72 hours caused the following: (1) significant increase of cell proliferation (CP) (CP24h =20.3%, CP48h =54.5% and CP72h =52.4%); (2) significant increase of cell population at S phase; (3) significant increase of autophagy demonstrated by (a) detection of AVOs; (b) LC3 IF positivity; (c) LC3 over expression (expression ratio24h =1.4, expression ratio48h =6.2 and expression ratio72h =11.4); (d) CQ increased LC3-II accumulation; (e) moreover, we demonstrated that the induction of autophagy by TCDD in PC3 cells was accompanied by an increase in the mRNA levels of certain genes that are present in different subnetworks composing the autophagy interactive network, such as: PIK3C3, BECN-1, AMBRA1, MAP1LC3B, ATG4A, ATG4C, ATG5, ATG7, ATG10, ATG16L1, GABARAPL1, PRKAA, WIPI-1. Also, we showed that TCDD negatively influenced genes that are autophagy inhibitor, such as: AKT1 and BCL2. These data suggested a multiple effect of TCDD on autophagy machinery. Furthermore, we revealed that TCDD exposure upregulated TNF whose high levels have been related to prostate cancer progression via stimulation of proliferation, survival of malignant cells and increased resistance to chemotherapeutic agents.
From the 1980s, several illegal and uncontrolled sites of urban, toxic, and industrial waste disposal have been active in Campania region, Southern Italy, with the highest concentration reached in Naples and Caserta provinces. In 1994, Campania region is under a declared State of Emergency, because of the saturation of regional waste treatment facilities. There are growing national and international evidences that the accumulation of waste, illegal and legal, urban and industrial, has contaminated soil, water, and the air with a range of toxic pollutants including dioxins. A high correlation between incidences of cancer, respiratory illnesses, and genetic malformations and the presence of industrial and toxic waste landfills was also found. In Spring 2002, a dioxin emergency emerged in this region, as result of National Control Program of Residues in Foodstuff carried out by Health Minister in 2001, indeed, levels of TCDD exceeding the European Union tolerance were detected in dairy products and milk from cow and water buffalo, raised on in some areas of Campania Region. Cancer mortality descriptive studies demonstrated an increase of incidents rates of the prostate cancer in the same areas where dioxin levels were elevated. Furthermore, studies performed in animal model suggested that TCDD exposure is associated with abnormal prostate development, altered prostate pathology and increase susceptibility to prostate cancer. The administration of TCDD to a variety of cultured cells may alter their ability to proliferate and die. In a previous study we demonstrated that TCDD induced proliferation in Madin-Darby Bovine Kidney (MDBK), an epithelial cell line, in which analysis of MDBK cell morphology revealed some alterations in a large number of exposed cells, where neither signs of apoptosis nor necrosis were detected, but we found that TCDD activated cell death with autophagy. 
Herein, TCDD exposure in PC3, a cancer cell line, induced no signs of cell death. So, taken together, our results support the idea that TCDD, may induce the progression of prostate cancer enhancing cell proliferation, inducing autophagy, deregulating the expression of genes related to the autophagy machinery, and upregulating TNF resulting in an increased risk for both animal and human health.