Placental glutathione S-transferase correlates with cellular proliferation during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Abstract

Taking into consideration that glutatione S-transferase (GST) and cellular proliferation play a crucial role during carcinogenesis, the goal of this study was to investigate the expression of placental GST, called GST-P, and proliferating cellular nuclear antigen (PCNA) by means of immunohistochemistry during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). This is a useful model for studying oral squamous cell carcinoma phase by phase. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution by drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. GST-P positive foci were detected in non-neoplastic oral cells at 4 weeks of 4NQO administration. In the same way, GST-P positive cells were detected in pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks-treatment, respectively. None of the control animals expressed GST-P positive cells. Regarding cellular proliferation, PCNA positive nuclei were higher at 12 and 20 weeks following 4NQO exposure (p<0.05) when compared to negative control. These results suggest that the expression of GST-P is correlated with cellular proliferation, in which GST-P is associated with risk and progression of oral cancer, whereas PCNA is closely involved during neoplastic conversion.

Introduction

Squamous cell carcinoma is the most common malignancy that affects the human oral cavity (Sakai and Tsuchida, 1992). Despite recent advances in therapeutic modalities, the prognosis of patients with oral squamous cell carcinoma has not been improved significantly in the last decades (Ribeiro et al., 2004). Herein, it is desirable to examine the precise pathobiological mechanisms involved in oral tumorigenesis in order to introduce reliable biomarkers to prevent oral squamous cell carcinomas. The most used animal models in oral cancer research are the hamster buccal pouch by fat-soluble 7,12 dimethylbenzanthracene (DMBA), and the rat tongue by water-soluble 4-nitroquinoline 1-oxide (4NQO). Considering that one of the most important routes of oral carcinogens is through liquid containing water-soluble carcinogens, 4NQO is well suited in examining the role of xenobiotics in experimental oral carcinogenesis (Tanaka et al., 2002). Based on the multi-step process of carcinogenesis characterized by initiation, promotion and tumor progression, chronic administration of 4NQO in drinking water simulates rat tongue carcinogenesis like human counterpart (Ohne et al., 1985; Nishimura, 1999; Niwa et al., 2001; Okazaki et al., 2002; Vered et al., 2003; Ribeiro et al., 2005).

Glutatione S-transferases (GSTs) are a family of enzymes involved in detoxification of xenobiotics. GSTs exist as homo- or heterodimers and have been grouped into at least seven distinct classes (Hayes and Pulford, 1995; Strange et al., 2000). The main function of GSTs is to catalyze the conjugation of glutathione to an electrophilic site of a broad range of potentially toxic and carcinogenic compounds, thereby making such compounds less biologically active and enabling their excretion (Hayes and Pulford, 1995). Placental GST, called GST-P, is the main isoform in normal placental tissue and comprises 67% of the total GST concentration in this phase (Zusterzeel et al., 1999). During development, GST-P decreases in concentration and is absent in adult tissues (Fatemi et al., 2006). Interestingly, GST-P-positive foci have been detected in adult tissues during medium-term hepatocarcinogenesis assay being regarded a suitable biomaker for early detection of liver neoplasms (Fonseca et al., 2005; Kitano et al., 2006; Uda et al., 2006). However, the real significance of GST-P positive cells during rat tongue carcinogenesis induced by 4NQO is fairly limited in literature (Li, 1999).

It has been well established that tumor cell proliferation activity plays an important role in tumorigenesis (Tumuluri et al., 2004). Among proliferating cellular markers, PCNA is a DNA polymerase delta auxiliary protein of 36 kDa, which is closely related to the replication of DNA and is indispensable to cell proliferation. Since the PCNA level increases rapidly in mid-G1, remains elevated throughout the S phase, and begins to decrease from G2/M to G1 (Linden et al., 1992), a decrease in the number of PCNA-positive cells reflects a decrease in S phase cells and thus a reduced proliferative activity. To our knowledge, there are no studies that addressed concomitant expression of GST-P and PCNA during experimental oral carcinogenesis so far. As a result and because of inapropriate evidence, the present study was undertaken to investigate the expression of GST-P positive foci and PCNA-positive cells on 4NQO-induced rat tongue carcinogenesis.