APÉNDICE PROCESOS DE LA MATEMÁTICA
Q: símbolo con el que se representa el conjunto de los números racionales.
Prostate cancer is the most common form of cancer affecting men in the western countries and the second leading cause of death after lung cancer. In the UK, more than 35,000 men were diagnosed with prostate cancer and around 10,000 men die from prostate cancer each year. Current treatment of prostate cancer mainly relies on a combination of androgen ablation, radiotherapy and chemotherapy. However, tumors often return with more aggressive hormone independent form. So it is important to improve the understanding of molecular mechanisms involved in prostate cancer malignant progression, which may lead to new therapies.
The risk factors for prostate cancer include age, ethnicity, family history and diet. It has been reported that prostate cancer onset has been attributed to a variety of diet factors ranging from lack of selenium supplementation [183] to vitamin D [184] and high dairy fat intake was also associated with increased risk of prostate cancer [185-187]. A long- term study demonstrated that high blood levels of trans fatty acids were associated with an increased prostate cancer risk [188].
This thesis investigated the relationship between the tumourigenicity-promoting function of C-FABP and its fatty acid-binding ability. Firstly, transfectant cells with different abilities of binding to fatty acids have been established and the tumourigenicity of these transfectants were compared to assess whether the reduced fatty acid-binding capability is associated with the decreased ability of producing tumours. Like other
FABP family proteins, C-FABP binds to fatty acids through a binding motif which consists of 3 key amino acids (Arg109, Arg129, and Tyr131). Thus we used site-directed
mutagenesis to convert Arg109or Arg109 and Arg129 into Ala109 or Ala109 and Ala129 to
generate two mutant cDNAs and transfected them and wild type cDNA into the LNCaP cells to generate transfectant cell lines. Realtime PCR measurement on C-FABP mRNA showed that the C-FABP mRNA level in LNCaP-WT cells was increased by 137.5-fold comparing with the control cells. Western blot also confirmed the overexpression of C- FABP in LNCaP-WT cells. From the results obtained by our previous studies, it was demonstrated that the biological function of C-FABP is promote malignant progression of cancer cells. In this study, the effect of high level increment of C-FABP expression on cell malignant properties such as proliferation rate, invasiveness and anchorage-independent growth as an indication of tumourigenecicy, was investigated. It is also showed that the mutant C-FABP mRNA levels expressed respectively in LNCaP-R109A and LNCaP-R109/129A cells were also increased to the levels similar to that in LNCaP-WT cells. Thus these transfectants are ideal cell lines used for comparing biological functions exerted by wild type and mutant C-FABPs.
As mentioned previously, fatty acid uptake is regulated by certain FABPs in LNCaP cells [175]. Quantitative assessment showed that the relative levels of fatty acid uptakes in LNCaP-WT, LNCaP-R109A and LNCaP-R109/129A were increased 3.1-, 1.6- and 1.4- fold of that detected in control cells. In another word, in LNCaP-WT, LNCaP-R109A and LNCaP-R109/129A cell lines, 210%, 60% and 40% more fatty acid uptakes occurred comparing to that in the control cells. This result showed that increased wild type C-FABP expression by 137.5 times in mRNA level can result in an
increment in fatty acid uptake to 3.1- time of that in control; whereas one mutation to the 3 key amino acids in fatty acid binding motif of C-FABP deprived its fatty acid up-taking ability by 72% (1-60%÷210%). Similarly, two mutations to C-FABP caused 81% reduction in its fatty acid up-taking ability. These results suggested that the structural integrity of its fatty acid-binding motif is important for the ability of C-FABP to bind and to transport fatty acids into these transfectant cells and changing one key amino acid in this part can result losing most fatty acid-binding and transporting ability.
The biological properties of transfected cells with high up-taking level of fatty acids seem to be much more aggressive when compared to those of control cells, LNCaP-V. When tested in in vitro assays, the proliferation rate, invasiveness and tumorigenicity of LNCaP-WT cells with high level of wild type C-FABP expression were significantly enhanced. However these properties in LNCaP-R109A and LNCaP-R109/129A cells which express high level of mutated C-FABP, stayed at similar level as those in LNCaP-V cells. The results obtained from in vivo assay for tumorigenesis have same the pattern as in vitro assays. When inoculated with LNCaP-WT cells, 88.9% nude mice developed tumors. On the other hand, only 50%, 37.5% and 37.5% of the mice inoculated with LNCaP-R109A, LNCaP-R109/129A and LNCaP-V cells respectively yielded tumuors. The average tumor weight produced in LNCaP-WT group was also increased more than 13-times to 614.3±192mg compared that in LNCaP-V group (46.7±15.3mg). These data showed that increase the ability of up-taking fatty acids in LNCaP cells by elevate C-FABP expression was able to promote the proliferation, invasiveness and tumorigenicity both in vitro and in vivo. It has also been demonstrated that the function of C-FABP to stimulate tumourigenicity was reduced when its Page | 177
up-taking fatty acids capability was decreased. Taken together, these findings indicated that the increased up-taking of fatty acids played an important role in tumourigenicity- promotion. Our previous work demonstrated that suppressing expression of C-FABP in the highly malignant prostate cancer cell line PC-3M has inhibited its ability of forming tumours in nude mouse [189, 190]. The results of the current and previous work further confirmed that C-FABP promotes malignant progression of prostate cancer depending on its binding and transporting intracellular fatty acids capacity.
Angiogenesis plays an important role in growth, malignant progression and metastasis by promoting endothelial cell proliferation, invasion and capillary differentiation [191- 193]. In addition, Weidner et.al has reported that the pathological surrogate for angiogenesis (microvessel density) is correlated with malignancy of prostate cancer [130]. The current studies showed that angiogenesis-associated genes such as VEGF significantly increased the prostate cancer risk. The results in chapter 5 showed that increased expression of biologically active VEGF was detected in wild type C-FABP transfectants, but not in the mutant types of the transfectant cells. These results suggested that increased VEGF expression may be caused by wild type C-FABP and that the ability of C-FABP to increase VEGF depends on its fatty acid-binding ability. Fatty acids appeared to play important roles. Fatty acids were identified as signalling molecules [194] which may be recognised by their nuclear peroxisome proliferator- activated receptors (PPARs), particularly PPARβ/δ [32].
According to the results in this study and those reported by some other groups, it is reasonable to hypothesis that there may be fatty acid-initiated signalling pathways
leading to malignant progression in prostate cancer cells. The route of these pathways may be as following: the elevated expression of C-FABP may give rise to an increased total uptake of fatty acids and enhanced the possible fatty acid signalling activity. Thus, it may be possible that excessive levels of free fatty acids are transported by C-FABP into the nucleus of the cancer cells to activate some as yet unknown mechanisms which might initiate a chain of molecular reactions resulted in promoting cancer growth and expansion. One of such actions regulated through this unknown mechanism is to up-regulate some important down-stream “cancer-promoting” genes, such as VEGF [164, 189], and hence contribute to carcinogenesis. Thus C-FABP which acted as an intracellular fatty acids transporter may play a key role during this process. A schematic hypothesis for the involvement of C-FABP in cancer malignant progression is shown in Figure 8.1.
Figure 8.1 Model for possible C-FABP signaling pathways in cancer malignant progression
As a first step of testing this hypothesis, we investigated the relationship between the tumourigenicity-promoting function of C-FABP and its ability of binding to and
transporting fatty acids in this study and demonstrated that fatty acid-binding and transporting ability is essential for C-FABP to promote tumor growth and expansion. To fully establish this proposed route of fatty acids signaling pathways, the future work should be performed to study the detailed molecular mechanisms on how VEGF is up-regulated? Effort is also needed to study whether there are any other cancer- promoting molecules up-regulated by this mechanism?
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