Introduction

Epidemiology of cancer

Current cancer-control strategies do not seem work effectively according to the Lancet (Cavalli, 2013). In 2010 an estimation of 8 million people died from cancer, a 38% rise on the annual cancer death toll compared to 1990 (Lozano et al., 2013). A dramatic increase in cancer incidence has occurred in low- and middle-income countries undergoing rapid socioeconomic transition (Soerjomataram et al., 2012), such as China. By 2030, it is predicted that almost two-thirds of cancer cases will occur in these countries (Bray et al., 2012) and more than 13 million deaths from cancer are estimated by WHO (Globocan 2008, IARC, 2010). Cancer is a leading cause of death and a major health concern worldwide. The main types of cancer are lung, stomach, liver, colorectal, breast, prostate and cervical cancer (Cancer fact sheet 2013, World Health Organisation). Cancer is not a single disease but many diseases. Transformation of a normal cell into a cancer cell is a multistage process including changes such as oncogene activation, loss of telomerase and aneuploidy induction (Bergers & Benjamin, 2003). Tumorigenesis is a long term process that cells may generate mutations as a result of exposure to external carcinogens, which include physical carcinogens, such as ultraviolet and ionizing radiation; chemical carcinogens, such as asbestos and tobacco; and biological carcinogens, such as viruses and bacteria. In addition, the incidence of cancer rises with age in that overall risk factors accumulate with age whereas cellular repair systems attenuate (Cancer fact sheet N°297, WHO, 2013). A group of key risk factors attributable to more than 30% of cancer death exist that can be modified or avoided.

 These include: tobacco use, being overweight or obese, unhealthy diet with low fruit and vegetable intake, lack of physical activity, alcohol use, sexually transmitted human papilloma virus (HPV)-infection, urban air pollution, and 2 indoor smoke from household use of solid fuels. Effective strategies to reduce cancer incidence and mortality are: avoidance of these risk factors; early detection, diagnosis and treatment; and screening of asymptomatic population at risk (WHO, 2013). 

Polyamines

History of polyamines

Spermine was the first polyamine discovered in human semen by Van Leeuwenhoek in 1678. In the late 1800s, putrescine was identified in microbes and spermidine was eventually found in early twentieth century (Gerner & Meyskens Jr, 2004). The first research article “polyamines and cancer cells” was published by the Proceedings of the National Academy of Japan in 1958 (Criss, 2003). Since then, over 85,000 papers have been published on polyamines in the past 55 years. Polyamines have undoubtedly contributed to the knowledge of biological sciences and are still doing so as the mystery of their molecular functions has not been fully elucidated yet, despite the huge progress has been achieved thus far. 

Removal of Tet from culture to induce SSAT expression

Tet was very difficult to wash off. In order to remove completely, Tet concentration in medium was decreased to 100 ng/ml 24 h prior to passage. On the day of passage, cell culture was washed twice in PBS before trypsinisation. After the cells detached, Tet-free culture medium was added to neutralise trypsin. 

The cell pellet was resuspended in Tetfree culture medium after centrifugation at 200 g for 3 min. SSAT+ (Tet-off) cells were plated first and then SSAT- (Tet-on) cells. After 24 h growth, medium was replaced with fresh medium to ensure complete wash-off of Tet. 

Aseptic techniques

Standard good laboratory practice is always essential. Cell culture procedures were carried out aseptically in a Class ІІ air-flow biohazard hood (microbiological safety cabinet). Gloves were worn all the time. The work area of cabinet was wiped with 70% (v/v) ethanol before and after use. Items for cell culture use in the cabinet must be either sprayed with 70% ethanol or autoclaved prior to use. Solutions were usually sterile filtered using a 0.22 µm syringe filter prior to use on cells. Cell culture waste was autoclaved at 121°C for 20 min before disposal. 

Maintenance and subculture of anchorage dependent cells (Passage)

LNCaP and LNG53 (human SSAT cDNA transfected LNCaP) cell lines were cultured routinely in a T75 cm2 cell culture flask containing 15 ml culture medium at 37°C in a humid incubator supplemented with 5% CO2. Cells were passaged when they became 80% confluent in the culture flask. Confluence was defined as maximal cell growth without spare space for cell spreading. Reagents were warmed to 37°C in a water-bath before use. Used medium was discarded from the flask to a waste bottle. Cells were washed with 10 ml PBS and then 2-3 ml trypsin-EDTA for LNCaP or 0.25% (v/v) trypsin for LNG53 was added. Cells were further incubated for 3-5 min until detached (gently tapping the flask can help the cells detach). 

This method was developed to be more sensitive and to allow for the measurement of acetylpolyamines (Section 2.2.13). It is equally important to characterise the effect of SSAT alteration on total polyamine content since SSAT is responsible for polyamine degradation. In general, total polyamine content of SSAT+ cells appeared higher than that of the other two cell types and it was significantly different at 96 h comparing to WT, although the difference observed was not significant between SSATand SSAT+ (Fig. 3.13). This implies that overexpression of SSAT activity tends to increase total polyamine content and individual polyamine content can also change dramatically. WT and SSAThad a low content of N1 -acetylspermidine whereas N1 -acetylspermidine in SSAT+ cells was increased significantly (4-7 fold), which confirmed that SSAT activity was induced (Fig. 3.14a). Likewise, N1 -acetylspermine presented the same pattern (Fig. 3.14b), but the concentration was about 10 fold lower than N1 -acetylspermidine in SSAT+ cells. Putrescine content was also significantly higher (4-8 fold) in SSAT+ than the other two cell types (Fig. 3.14c). The increase in putrescine was consistent with the enhanced ODC activity (Fig. 3.7) and putrescine being the final product from the retroconversion pathway. Spermidine tended to decline after 72 h in all the cells (Fig. 3.14d), which was probably because the proliferation rate was gradually slowed down as the cells were reaching high cell density by 72 h growth in a 60 mm culture dish. 

 The Tet-off system is reversible and controlled at the level of Sat1 gene expression. Therefore, changes following the “Tet switch” in the polyamine pathway are a result of SSAT alteration. Additionally, WT LNCaP cells were included as a “reference control.” Thus, all the experimental results can be compared on the background of low (SSAT- ), basal (WT), and high (SSAT+ ) SSAT activity. The model system was established successfully at the level of Sat1 mRNA expression, SSAT protein and SSAT activity (Fig. 3.3, 3.4, & 3.6). This model system is simple to control and allows for the analyses of the effects of these changes in the polyamine system. A correlation between a decreased cell growth and an increased SSAT activity has been established by Libby, et al. (1991). In this study, a lower SSAT activity is correlated with a shorter cell generation time (48-96 h), and vice versa (Fig. 4.1). This is consistent with the study by Kee et al. (2004b) in which this SSAT transfected LNCaP cell model system was firstly published. The WT cells were not part of their study. Involvement of the WT cells demonstrates clearly the difference in cell growth between these cells. Similar correlations are found in MCF-7 human breast cancer (Vujcic et al., 2000), CHO Chinese ovarian cancer (McCloskey et al., 1999), and NCI-H82 small-cell lung carcinoma (MurrayStewart et al., 2003) SSAT transfected cell lines.In addition, a large number of studies using polyamine analogues, BENSpm (or DENSpm) in particular, also show a growth inhibition associated with a superinduction of SSAT in some cell lines (Libby et al., 1991; Vujcic et al., 2000; Murray-Stewart et al., 2003; Pegg, 2008). 

 However, in our study the polyamine analogue CPENSpm does not affect cell growth but is a superinducer of SSAT. Accordingly, it is likely that an increase of SSAT activity appears to be a common feature involved in growth inhibition in a variety of tumour cell lines. Since SSAT is responsible for polyamine degradation, the growth inhibition is connected somehow to an alteration of intracellular polyamine pools. Alteration of SSAT activity exerts a dramatic impact on the individual polyamine concentrations. Polyamine metabolism is a pathway that is tightly regulated by biosynthesis, degradation, uptake and export. When one parameter is altered in the pathway this gives rise to compensatory changes in other parameters. All of the changes observed in the polyamine system in this study, such as increased ODC, APAO and SMO, are aiming to maintain the intracellular polyamine concentrations in the appropriate range for cell survival and growth. As a result, the total polyamine content in SSAT+ cells does not decrease, which is consistent with the study of Kee et al. (2004b). Instead, both total intracellular and extracellular polyamine pools in SSAT+ cells tend to increase (Fig. 3.13 & Fig. 3.40a-f). This is mainly attributable to the accumulation of N1 - acetylspermidine inside and outside of the cells (Fig. 3.35e & f). Acetylation of spermidine by SSAT to form N1 -acetylspermidine and its subsequent efflux are the main mechanisms lowering intracellular polyamines pools. Additionally, N1 ,N12 -diacetylspermine (Fig. 3.33d) acetylated from N1 -acetylspermine, and putrescine (Fig. 3.37f) synthesised from ODC and the retroconversion pathway, are the second most abundant polyamines exported as a result of increased SSAT activity.

 Thus, polyamine acetylation is the first essential step to reduce polyamine content instead of direct export of spermidine and spermine out of the cell, despite that a small amount of spermidine can be exported without acetylation. A depletion of spermidine and spermine leads to an arrest of cell growth or cell death. Spermidine and spermine are degraded although total polyamine content is maintained or even tends to increase, while SSAT is induced. SSAT overexpression leads to a fall of spermidine and spermine that can result in DNA damage and subsequent decreased cell proliferation (Zahedi et al., 2007). The loss of spermine (significant) and spermidine (not significant) in SSAT+ and the accumulation of spermidine and spermine in SSAT - cells are likely to cause the discrepancy in cell growth in the current study, for spermidine and spermine are critical in the modulation of functions of RNA, DNA and of protein synthesis (Igarashi & Kashiwagi, 2000). For example, spermidine is required in hypusine modification that is essential for eIF5A activity, which is critical for eukaryotic cell growth and survival (Lee et al., 2011). Sufficient amounts of intracellular spermidine and spermine are essential for cell survival or growth. The accumulation of spermidine and spermine due to insufficient degradation by low SSAT activity can stimulate cell proliferation. This may explain why SSATcells have the highest proliferation rate. Spermine is absent from the culture medium of all the cell types also indicates its essential nature as a growth factor required by the cells. Given that spermine is the major polyamine in the prostate gland and LNCaP cells (Schipper et al., 2003; McCloskey et al., 2000), the loss of spermine could attenuate cell growth. 

A dynamic equilibrium state of polyamine metabolism is supposed to be reached under physiological conditions. When the equilibrium is disturbed such as by SSAT alteration, the priority for the cells is to survive rather than proliferate. Thus, proliferation rate would decrease. SSAT induced decrease of intracellular spermine can be growth inhibitory to the prostate cancer cells. In terms of spermidine and spermine content, there was almost no difference between SSAT+ and WT (Fig. 3.14d & e), but WT cells grew faster than SSAT+ cells. This suggests that the accumulation of N1 -acetylspermidine, N1 -acetylspermine, and/or putrescine is likely to be growth inhibitory.Co-treatments of DFMO and MDL72527 partially restored SSAT+ cell growth (Fig. 3.43c). This is associated with the decreased putrescine but increased spermine, suggesting that putrescine accumulation is growth inhibitory and spermine restoration is growth stimulating. In addition, growth inhibition of lymphocytes by MGBG is thought to be caused by the decrease of spermidine and spermine but not by the accumulation of putrescine, because the growth inhibition can be reversed by exogenously added spermidine and spermine (Fillingame et al., 1975). With regard to N1 - acetylspermidine in the co-treatments in SSAT+ cells, the lack of N1 -acetylspermidine accumulation but accumulation of N1 -acetylspermine are due to the decreased spermidine and increased spermine.  On the other hand, the accumulation of the N1 - acetylpolyamines is not growth inhibitory as the growth of SSATand WT cells were not inhibited by the accumulation of the N1 -acetylpolyamines in the presence of MDL 72527.

 In vivo studies of SSAT transgenic mice presented a typical hairless phenotype that is thought to be due to the toxic accumulation of putrescine in hair follicles, which is phenotypically similar to ODC overexpressing transgenic mice (Pietilä et al., 1997). However, the exogenous addition of N1 -acetylspermidine or putrescine (≤ 10 µM) to the cell culture medium did not seem to inhibit the growth of both SSATand WT cells. This lack of growth inhibition might be also due to a failure of uptake of these polyamines into the SSATand WT cells. Similarly, an addition of putrescine (5 mM) to the culture medium of the tolerant CHO cells did not result in a toxic intracellular because of the upregulated export and decreased uptake of putrescine (Pastorian & Byus, 1997). On the contrary, ODC overexpressing CHO cells have been found to undergo cell death by apoptosis due to an over-accumulation of intracellular putrescine (Takao et al., 2006), indicating the toxicity of putrescine accumulation. N 1 ,N12 -diacetylspermine is known as a tumour marker found in the urine of a range of cancer patients. Although its concentration is very low, it is more reliable and specific than monoacetylpolyamines such as N1 -acetylspermidine and N8 -acetylspermidine (Kawakita & Hiramatsu, 2006). However, the SSAT activity is not measured in their study. Wallace et al. (2000) reported that an increase in SSAT activity in the tumour tissue of breast cancer comparing with normal tissue. This implies an increase of acetylpolyamines by SSAT in tumour tissue. What is consistent with our findings is that N1 ,N12 - diacetylspermine is present only in the culture medium. 

It is supposed to be exported out of the cell as a result of increased SSAT activity since it is only found intracellularly in SSAT+ cells, at least in vitro. It is believed that N 1 ,N12 -diacetylspermine can serve as a prognostic indicator and marker for recurrence of prostate and colon cancers, because it rises rapidly concomitant with the recurrence (Kawakita et al., 2004). If this is the  case, an increase of SSAT would be a sign of increased carcinogenesis or relapse, which is consistent with the findings in the SSAT transgenic Apcmin/+ mice (Tucker et al., 2005). However, N1 ,N12 -diacetylspermine content was not determined in that study. Polyamine acetylation by SSAT is closely connected to polyamine export. Polyamine export is known as a selective and regulated process dependent on the growth status of the cell (Wallace et al., 2003). Efflux is the major mechanism to decrease excess intracellular polyamines and prevent their accumulation. With the increase of SSAT, polyamine efflux is greatly increased. N 1 -acetylspermidine and putrescine consist of the major part of the increased polyamines in the culture medium of SSAT+ cells, which is consistent with the findings by Wallace & Mackarel (1998). It confirms that N 1 - acetylspermidine and putrescine are the predominant forms of polyamines for export and polyamines are required to be metabolised (i.e. acetylated) before efflux (Wallace et al., 2003). This might suggest that polyamine transport is a selective process determined by the requirements of the cell. The compensatory increase in ODC is also found in SSAT transgenic livers (Pietilä et al., 1997), transgenic fibroblasts (Alhonen et al., 1998) and SSAT-transfected CHO cells (McCloskey et al., 1999).