Gallbladder carcinomas: An immunoprognostic evaluation of P53, Bcl-2, CEA and alpha-fetoprotein

Cancer of the gallbladder is the fifth most common can­cer of the digestive tract. The mean five-year survival rate has remained at 1% despite surgical intervention; this is mainly attributed to its late presentation, with extensive lo­cal spread and concomitant invasion of the liver. It is now accepted that most cases of invasive gallbladder cancers arise from precursor lesions of intestinal metaplasia, dysplasia and carcinoma in situ lesions. The adenoma-cancer sequence is recognized in the minority of cases. In this study, the expres­sion of P53, Bcl-2, carcinoembryonic antigen (CEA) and alpha-fetoprotein was evaluated in both the in situ and inva­sive components of the tumour, and the results were corre­lated with their mean survival.

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Similarly to other genes polymorphisms, significant differ­ences exist in VDR polymorphisms distribution among differ­ent ethnic groups. A possible explanation to polymorphism generation phenomena is DNA damage events in an ancient small population that grow up in frequency becoming poly­morphisms in modern populations. VDR haplotypes distribu­tion reflects out-of-Africa evolution theory that describe hu­man populations origin and dispersion around the world, where gene-environment interactions favours the survival of some allelic variants. Old polymorphisms might show large population/ethnic variability (FokI), while new ones are likely characterized by small population variability (Cdx2). Assum­ing that a polymorphism should have the same functional ef­fect in different ethnic groups, different allele frequencies across these groups may explain differences in the incidence of pathologies and variability on drug response among them (i.e. Asians seem to be more vitamin D-sensitive while Cau­casians appear more oestrogen-sensitive than other ethnic groups). In the case of non-functional polymorphisms, fre­quencies of these markers are very different among ethnic groups. It is also difficult to understand consequences of eth­nic allele variation in this case, because of environmental fac­tors interference, such as diet and physical exercise. This emphasises that an haplotypes map of VDR polymorphisms in different ethnic groups is necessary.

Functionality of VDR polymorphisms

Functional studies are needed to determinate the way certain haplotypes in a candidate gene affects protein function. Com­pared to the larger number of genetic association studies, there has been little published on the mechanism through which VDR gene polymorphisms might influence VDR receptor functionality.

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Vitamin D endocrine system positively affects infection, autoim­mune diseases, tolerance in transplantation and this mainly de­rives from prodifferentiating effects on monocyte- macrophages, antigen-presenting cells, dendritic cells (DC) and lymphocytes as evidenced from in vivo and in vitro studies (Figure 4).

One of the mechanisms mediating vitamin D function in resis­tance to infections is the already mentioned induction of C/EBPP which enhances monocyte differentiation to macrophage, immune function, host defence against bacterial infection and tumour cell growth and production of IL-12, the cytokine inducing Th1 response. On the other hand y-interferon is a potent inducer of 1 a-hydroxylase in

macrophage thus increasing vitamin D local production through a C/EBPp-mediated mechanism. Local macrophage-pro- duced vitamin D is also an inducer of T cell response to cuta­neous antigens in vivo, including CD4-Th2 cell-mediated and mucosal antibody responses. In contrast to these stimu­latory effects on monocyte-macrophages, vitamin D is an im- munosuppressor for lymphocytes. This effect is due to a vitamin D/VDR-mediated inhibition of expression of cytokines involved in T cell functions, including IL-2. Vitamin D plays also an important role in the establishment and mainte­nance of immunological self-tolerance as observed in studies on animal models demonstrating a vitamin D-induced inhibition of disease induction in experimental autoimmune encephalomyelitis, thyroiditis, insulin-dependent diabetes melli- tus, inflammatory bowel disease, systemic lupus erythemato- sus and both collagen-induced arthritis and Lime arthritis. Finally vitamin D inhibits rejection of transplanted tissue probably through a VDR-mediated mechanism involving TGFp/Smad3 interactions. In conclusion vitamin D seems to be a modulator of the immune response mainly act­ing through a paracrine loop which may block inflammation and/or modulate the differentiation of activated CD4 T cells as well as the suppressor T cell function.

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Vitamin D acts in parathyroid tissue through its binding to VDR and consequent regulation of gene transcription and a VDRE was demonstrated in the promoter region of the PTH gene. Thus 1,25(OH)2D3 exerts a negative feedback on PTH production in response to PTH-dependent activation of calcium mobilization from kidney and bone, through a VDR-mediated silencing of PTH gene transcription. Moreover 1,25(OH)₂D₃ regulates parathyroid level of VDR and its response to calcium. The first effect is due to an increase in VDR mRNA level, possibly secondary to serum calcium increase, as well as to a ligand-dependent VDR protection from proteosomal degradation. The second one directly involves VDR-mediated regulation of calcium sensing receptor (CaSR) gene transcription as evoked by the presence of two VDREs in its promoter region. The other important effect of 1,25(OH)₂D₃ on parathyroid glands involves cell growth regulation and is described in other chapter.

Effects on the kidney

One of the major effect of vitamin D in the kidney is its own homeostasis through inhibition of 1 a-hydroxylase and stimula­tion of 24-hydroxylase expression as well as through induction of megalin expression in the proximal tubule. Moreover it stimulates renal calcium reabsorption and enhances calbindin expression, accelerating PTH-dependent calcium transport in the distal tubule (Figure 4). Finally vitamin D/VDR acts on TRPV5 promoter to increase its mRNA and protein levels which is an important actor in vitamin D-mediated calcium reabsorption.

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Effects on intestinal calcium

The role of vitamin D in intestinal absorption of calcium is well known and the vitamin D endocrine system has been finally identified as the agent that stimulates intestinal calcium ab­sorption to meet the needs of the skeleton (Figure 4). Ep­ithelial calcium channel TRPV6 (CaT1 or EcaC2) is necessary for calcium uptake together with TRPV5 (EcaC1). Then cal- bindin D shuttles the ion across the cell and finally the plasma membrane Ca²⁺ ATPase (PMCA1b), and the Na⁺/Ca²⁺ ex­changer (NCX1) deliver it into the bloodstream. TRPV5 and TRPV6 expression levels depend on VDR regulation as demonstrated by their reduction in VDR-null mice and their in­duction by calcitriol supplementation in wild-type mice. These two channels confer high Ca²⁺ selectivity and negative feedback regulation to intestinal Ca²⁺ influx, strictly resembling that in native distal renal cells. Vitamin D has a clear role even in stimulating intestinal absorption of phosphate which is another active calcium transport mechanism but which appears to be completely independent from the direct one. VDR-mediated mechanism to maintain the Ca PO₄ ion product seems to primary involve induction of phosphate translocating proteins in kidney and perhaps in intestine. Indeed the renal sodium-phosphate cotransporter-2 (NPT2) is a likely vitamin D- induced protein containing a VDRE in the promoter region. Another role of VDR in phosphate homeostasis is due to 1,25(OH)₂D₃ induction of PEX gene expression. PEX gene, which harbours a VDRE in its promoter region, is a phos­phate-regulating gene, showing high homologies to endopepti- dases located on the X-chromosome and postulated to be the proteolytic agent for inactivation of phosphatonins which in turn have phosphaturic action through potent inhibitory effects on NPT2 and 1 a-hydroxylase. From a mechanicistic point of view, intestinal active phosphate transport is stimulated by a vitamin D-induced increase of Na- PO4 cotransporter and plasma membrane fluidity of enterocytes. On the con­trary the mechanism that mediates phosphate transport through basolateral membrane is still unknown.

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Role of PTH

The parathyroid gland is the calcium-sensing organ in the body and in a few seconds it responds to even slight hypocal- caemia by secreting the 84-amino acid peptide hormone PTH. Its receptor is expressed in the nephron and in osteoblasts but not in intestine and osteoclasts for example. In the kid­ney PTH acts as a phosphate antireabsorptive agent causing a phosphate diuresis, activates 25-hydroxyvitamin D-1 a-hy- droxylase and inhibits 24-hydroxyvitamin D-1 a-hydoxylases

through cAMP increase thus causing an increment in plas­ma levels of active 1,25(OH)2D3 (Figure 4).

Role of vitamin D

Vitamin D initiates an active intestinal calcium transport in the small intestine. This action has the longer lifetime, mea­surable in days, while the other actions are shorter. The results of the action of PTH and 1,25(OH)₂D₃ is the mobiliza­tion of calcium by the skeleton into the plasma compartment and this is obtained by either osteoclast stimulation to resorb bone and to reverse calcium transport from bone fluid compart­ment to plasma (Figure 4). Even in the distal renal tubule PTH and 1,25(OH)₂D₃ act synergistically to reabsorb the last 1% of the filtered load of calcium into the plasma compart­ment. Interestingly, through the study of animal mod­els, defective for either 1,25(OH)₂D₃ or PTH, it has become ev­ident that the presence of both hormones is required for this system to operate in vivo although the precise mechanism of the interaction between them is still poorly understood. Fi­nally the rise in serum calcium level resets the sensing point of the calcium receptor and shuts down the secretion of PTH.

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