Monica Notarbartolo

Resistance to diverse apoptotic triggers in multidrug resistant HL60 cells and its possible relationship to the expression of P-glycoprotein, Fas and of the novel anti-apoptosis factors IAP (inhibitory of apoptosis proteins)

We studied the human HL60 leukemia cell line and its multidrug resistant (MDR) variant HL60R. In contrast to the HL60, HL60R showed an inability to undergo apoptosis from doxorubicin (Dox) or other different stimuli, including cisplatin, Fas ligation and serum withdrawal. HL60R cells lost surface Fas expression, but we found no evidence that Fas/FasL mediates the apoptotic effects of Dox in HL60. P-glycoprotein (P-gp) did not seem to play a major role as a specific inhibitor of apoptosis. In fact, the P-gp inhibitor verapamil reversed only partially the resistance to Dox-induced apoptosis of the MDR cells. In addition, it did not modify the rate of apoptosis induced from the other stimuli in the same cells. The expression of p53 or Bcl-2 was not different between HL60 and HL60R. However, in HL60R there was an increase in the mRNAs of inhibitory of apoptosis proteins (IAPs) like neuronal apoptosis inhibitory protein (NAIP), c-IAP-2 and survivin. Treatment with Dox or serum starvation strongly down-regulated X-linked IAP and survivin mRNAs in HL60. Cisplatin decreased NAIP and survivin mRNAs in the same cells. However, in HL60R the levels of these IAP mRNAs were much less affected by the treatments. These results support that IAPs may be involved in tumor resistance to chemotherapeutic drugs or other apoptotic agents.

Circulating IL-6 and sIL-6R in Patients with Hepatocellular Carcinoma

Abstract: Interleukin‐6 plays a central role in regulating the immune system, hematopoiesis, and acute phase reaction. It interacts with a receptor complex consisting of a specific ligand‐binding protein (IL‐6R, gp80) and a signal transduction protein (gp130). In this report, serum levels of IL‐6 and a soluble form of the interleukin‐6 receptor (sIL‐6R) were evaluated in patients with hepatocellular carcinoma. The correlation between IL‐6 and sIL‐6R values, the stage of hepatocellular carcinoma, and main liver function tests was also studied.

Truncated Form of β‐Catenin and Reduced Expression of Wild‐Type Catenins Feature HepG2 Human Liver Cancer Cells

GIUSEPPE CARRUBA, a,e MELCHIORRE CERVELLO,b MARIA D. MICELI,a ROSARIA FARRUGGIO,a MONICA NOTARBARTOLO,c LUCREZIA VIRRUSO,b LYDIA GIANNITRAPANI,d ROBERTO GAMBINO,b GIUSEPPE MONTALTO,d AND LUIGI CASTAGNETTAa,c aInstitute of Oncology, and dInstitute of Internal Medicine, University of Palermo, Palermo, Italy bInstitute of Developmental Biology, C.N.R., Palermo, Italy cExperimental Oncology, Palermo Branch of IST-Genoa, c/o M. Ascoli Cancer Hospital Center, Palermo, Italy

Expression of WISPs and of their novel alternative variants in human hepatocellular carcinoma cells.

Abstract: WISPs (Wnt‐induced secreted proteins) are members of the CCN (CTGF/Cyr61/Nov) family involved in fibrotic disorders and tumorigenesis. They have a typical structure composed of four conserved cysteine‐rich modular domains, but variants of CCN members lacking one or more modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. WISP genes were first described as downstream targets of the Wnt signaling pathway, which is frequently altered in human hepatocellular carcinoma (HCC). In the present study, WISP mRNA expression was analyzed by RT‐PCR in four human HCC cell lines (HepG2, HuH‐6, HuH‐7, HA22T/VGH). Our results show for the first time that WISP1, WISP1v, and WISP3 are expressed in HCC cell lines. Moreover, we identified two novel variants, generated by alternative splicing of WISP1 and WISP3, respectively, named WISP1Δex3‐4 and WISP3vL. Overall, our study suggests that WISP transcripts may have a role in the development of HCC, although further studies are necessary to clarify the relative importance of the expression of wild‐type WISPs, as well as of their novel variants, in this tumor type.

Induction of Apoptosis and Inhibition of Cell Growth in Human Hepatocellular Carcinoma Cells by COX‐2 Inhibitors

Abstract: The aim of the present study was to examine the effects of nonselective (indomethacin) and selective cyclooxygenase‐2 (COX‐2) inhibitors (NS‐398, nimesulide, and CAY10404) on cell growth, cell cycle distribution, and apoptosis in three human hepatocellular carcinoma cell lines (HepG2, HuH‐6, and HA22T/VGH) with different characteristics of differentiation and biological behavior. The four COX inhibitors showed a dose‐dependent growth‐inhibitory effect in all the cell lines. No substantial arrests in the progression of the cells through the cell cycle were observed after treatment of HuH‐6 or HA22T/VGH for 48 h with the various inhibitors. On the other hand, there were significant increases in apoptosis, with the highest effect of cell kill being seen after treatment with indomethacin, especially in HuH‐6. Our findings support the suggestion that selective or, perhaps more efficiently, nonselective COX‐2 inhibitors may have potential therapeutic effects in hepatocellular carcinoma. Further studies must be carried out to better determine the possible mechanisms of these effects.

Curcumin as a Possible Lead Compound against Hormone‐Independent, Multidrug‐Resistant Breast Cancer

We examine the possible evidence that the phytochemical curcumin may overcome resistance to hormonal and cytotoxic agents in breast cancer. We present our observations on MCF‐7R, a multidrug‐resistant (MDR) variant of the MCF‐7 breast cancer cell line. In contrast to MCF‐7, MCF‐7R lacks aromatase and estrogen receptor α (ERα) and overexpresses the multidrug transporter ABCB1 and the products of different genes implicated in cell proliferation and survival, like c‐IAP‐1, NAIP, survivin, and COX‐2. Nevertheless, in cytotoxicity and cell death induction assays, we found that the antitumor activity of curcumin is substantial both in MCF‐7 and in MCF‐7R. We elaborated the diketone system of curcumin into different analogues; the benzyloxime and the isoxazole and pyrazole heterocycles showed remarkable increases in the antitumor potency both in the parental and in the MDR MCF‐7 cells. Furthermore, curcumin or, more potently, the isoxazole analogue, produced early reductions in the amounts of relevant gene transcripts that were diverse (i.e., they were relative to Bcl‐2 and Bcl‐XL in MCF‐7 and the inhibitory of apoptosis proteins and COX‐2 in MCF‐7R) in the two cell lines. Thus, the two compounds exhibited the remarkable property of being able to modify their molecular activities according to the distinct characteristics of the parental and MDR cells. We discuss also how curcumin may (1) exert antitumor effects in breast cancer through ER‐dependent and ER‐independent mechanisms; and (2) act as a drug transporter‐mediated MDR reversal agent. Overall, the structure of curcumin may represent the basis for the development of new, effective anticancer agents in hormone‐independent MDR breast cancer.

Pharmacogenetic considerations for optimizing tacrolimus dosing in liver and kidney transplant patients

The introduction of tacrolimus in clinical practice has improved patient survival after organ transplant. However, despite the long use of tacrolimus in clinical practice, the best way to use this agent is still a matter of intense debate. The start of the genomic era has generated new research areas, such as pharmacogenetics, which studies the variability of drug response in relation to the genetic factors involved in the processes responsible for the pharmacokinetics and/or the action mechanism of a drug in the body. This variability seems to be correlated with the presence of genetic polymorphisms. Genotyping is an attractive option especially for the initiation of the dosing of tacrolimus; also, unlike phenotypic tests, the genotype is a stable characteristic that needs to be determined only once for any given gene. However, prospective clinical studies must show that genotype determination before transplantation allows for better use of a given drug and improves the safety and clinical efficacy of that medication. At present, research has been able to reliably show that the CYP3A5 genotype, but not the CYP3A4 or ABCB1 ones, can modify the pharmacokinetics of tacrolimus. However, it has not been possible to incontrovertibly show that the corresponding changes in the pharmacokinetic profile are linked with different patient outcomes regarding tacrolimus efficacy and toxicity. For these reasons, pharmacogenetics and individualized medicine remain a fascinating area for further study and may ultimately become the face of future medical practice and drug dosing.

Influence of CYP3A5 and ABCB1 gene polymorphisms and other factors on tacrolimus dosing in Caucasian liver and kidney transplant patients

Tacrolimus is a substrate of cytochrome P4503A (CYP3A) enzymes as well as of the drug transporter ABCB1. We have investigated the possible influence of CYP3A5 and ABCB1 single nucleotide polymorphisms (SNPs) and other factors (e.g. albumin, hematocrit and steroids) on tacrolimus blood levels achieved in a population of Caucasian liver (n=51) and kidney (n=50) transplant recipients. At 1, 3 and 6 months after transplantation, tacrolimus doses (mg/kg/day) and trough blood levels (C0) were recorded and the weight-adjusted tacrolimus dosage (mg/kg/day) was calculated. Polymerase chain reaction followed by restriction fragment length polymorphism analysis was used for genotyping CYP3A5*1 and *3 [6986A>G] as well as ABCB1 at exons 21 [2677G>T/A] and 26 [3435C>T] in both liver transplant donors and recipients and in kidney transplant recipients. Of the 152 subjects studied, 84.9% showed a CYP3A5*3/*3 genotype. The total frequency of the allelic variant *3 was 93%. For the G2677T/A and C3435T polymorphisms the total frequencies of the allelic variants T/A and T were 44.7 and 46.7%, respectively. At 1, 3 and 6 months after transplantation the dose-adjusted C0 levels were significantly lower in patients with one copy of the *1 allele compared to those homozygous for the *3 allele. In the case of liver transplant patients the tacrolimus dose requirements were dominantly influenced by the polymorphisms of the CYP3A5 gene in the donors. With regard to the ABCB1 SNPs, in general they did not show any appreciable influence on tacrolimus dosing requirements; however, kidney transplant recipients carrying the 2677T/A allele required significantly higher daily tacrolimus doses than subjects homozygous for the wild-type allele. Identification of CYP3A5 single nucleotide polymorphisms prior to transplantation could contribute to evaluate the appropriate initial dosage of tacrolimus in the patients.

Expression of IAPs and Alternative Splice Variants in Hepatocellular Carcinoma Tissues and Cells

Abstract: IAPs (inhibitors of apoptosis proteins) might have a major role in the apoptotic resistance that marks many cancers. The studies on IAPs in human HCC have focused on survivin or XIAP, indicating that their new or increased expression in this tumor is associated with a more unfavorable prognosis. The present results corroborate these findings, emphasizing the role that the coordinated expression of different IAPs and alternative splice variants might play in the adverse biology of hepatocellular carcinoma.

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells☆

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 microM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (delta4Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 microM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to delta4Ad. In either cell line, T transformation to delta4Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.