Paola Poma

Development and characterization of co-loaded curcumin/triazole-halloysite systems and evaluation of their potential anticancer activity.

Positively charged halloysite nanotubes functionalized with triazolium salts (f-HNT) were employed as a carrier for curcumin molecules delivery. The synthesis of these f-HNT new materials is described. Their interaction with curcumin was evaluated by means dynamic light scattering (DLS) and UV-vis spectroscopy in comparison with pristine unmodified HNT (p-HNT). The curcumin load into HNT was estimated by thermogravimetric analysis (TGA) measurements, while the morphology was investigated by scanning electron microscopy (SEM) techniques. Release of curcumin from f-HNT, at three different pH values, by means of UV-vis spectroscopy was also studied. Furthermore, different cancer cell lines were used to evaluate the potential cytotoxic effect of HNT at different concentrations and culture times. The results indicated that the f-HNT drug carrier system improves the solubility of curcumin in water, and that the drug-loaded f-HNT exerted cytotoxic effects against different cell lines.

Direct chemical grafted curcumin on halloysite nanotubes as dual-responsive prodrug for pharmacological applications.

Covalently functionalized halloysite nanotubes (HNTs) were successfully employed as dual-responsive nanocarriers for curcumin (Cur). Particularly, we synthesized HNT-Cur prodrug with a controlled curcumin release on dependence of both intracellular glutathione (GSH) and pH conditions. In order to obtain HNT-Cur produgs, halloysite was firstly functionalized with cysteamine through disulphide linkage. Afterwards, curcumin molecules were chemically conjugated to the amino end groups of halloysite via Schiffs base formation. The successful functionalization of halloysite was proved by thermogravimetric analysis, FT-IR spectroscopy, dynamic light scattering and scanning electron microscopy. Experimental data confirmed the presence of curcumin on HNT external surface. Moreover, we investigated the kinetics of curcumin release by UV-vis spectroscopy, which highlighted that HNT-Cur prodrug possesses dual stimuli-responsive ability upon exposure to GSH-rich or acidic environment. In vitro antiproliferative and antioxidant properties of HNT-Cur prodrug were studied with the aim to explore their potential applications in pharmaceutics. This work puts forward an efficient strategy to prepare halloysite based nanocarriers with controlled drug delivery capacity through direct chemical grafting with stimuli-responsive linkage.

Pharmaceutical properties of supramolecular assembly of co-loaded cardanol/triazole-halloysite systems.

Halloysite nanotubes were explored as drug carrier for cardanol, which is considered as a promising natural anticancer active species. To this aim, besides the pristine nanoclay, a chemical modification of the nanocarrier was performed by attaching triazolium salts with different hydrophobicity at the outer surface of the hollow nanotubes. The interaction between cardanol and nanotubes was highlighted in solution by HPLC. This method proved the loading of the drug into the nanotubes. The solid dried complexes formed by pristine and modified halloysite with the cardanol were characterized by IR spectroscopy, thermogravimetric analysis as well as water contact angle to evidence the structure, thermal properties and wettability of the obtained materials. The kinetics of cardanol release as well as cell viability experiments provided promising results that put forward a new strategy for potential applications of cardanol as active antiproliferative molecule and clay nanotubes as drug carrier.

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.

Lack of nucleophilic addition in the isoxazole and pyrazole diketone modified analogs of curcumin; implications for their antitumor and chemosensitizing activities

Curcumin (CUR) can be considered as a good lead compound for the design of new anticancer drugs. Further, structure-activity relationship studies may clarify the importance of the redox activities in the antitumor effects of the drug. We have elaborated the alpha,beta-unsaturated 1,3-diketone moiety of CUR into the isoxazole (ISO) and pyrazole (PYR) derivatives. These derivatives should be much less prone to nucleophilic addition than CUR and benzyl mercaptan addition analyses showed that indeed they do not form isolable conjugated products. When compared with CUR, ISO and PYR exhibited increased cell growth inhibitory and pro-apoptotic effects in liver cancer HA22T/VGH cells as well as in other tumor cell types; in contrast to CUR, the antitumor effects of ISO or PYR were not influenced by concomitant administration of N-acetylcysteine, as a source of -SH groups, or buthionine sulfoximine, as an inhibitor of glutathione synthesis. Further, treatment with CUR, but not with ISO or PYR, significantly decreased the content of reduced glutathione in the HA22T/VGH cells. Finally, ISO and PYR lacked the ability of the parent compound to sensitize the HA22T/VGH cells to cisplatin (CIS), an effect which appeared to occur through an interaction of CUR and CIS at the level of the -SH groups. Thus, the ability of interacting with cell thiols might not be requested for the more potent antitumor activities of new diketone modified CUR derivatives, which might rely on other mechanisms, though possibly devoid of chemosensitization capabilities.

Frequent Alteration of the Yin Yang 1/Raf-1 Kinase Inhibitory Protein Ratio in Hepatocellular Carcinoma

The transcription factor Yin Yang 1 (YY1) can favor several aspects of tumorigenesis. In turn, Raf-1 Kinase Inhibitor Protein (RKIP) inhibits the oncogenic activities of MAPK and NF-κB pathways and promotes drug-induced apoptosis. Mutual influences between YY1 and RKIP may exist, and there are already separate evidences that relevant increases in YY1 and reductions in RKIP occur in hepatocellular carcinoma (HCC). However, the levels of the two factors have never been concomitantly examined in HCC. We evaluated by RT-PCR the mRNA levels of YY1, YY1AP, RKIP, and survivin in 35 clinical HCCs (91% HCV-related), in their adjacent cirrhotic tissues and in 6 healthy livers. Immunohistochemical analyses were also performed. The ratio of YY1 to RKIP mRNA was constantly profoundly inverted in the tumors compared with the adjacent nontumoral tissues. A similar result occurred frequently at protein level. Hyperactivation of YY1 in tumors was corroborated by its nuclear localization and the finding that in the tumors there were also increases in YY1AP, a YY1 coactivator not expressed in normal liver, and in survivin, as a possible target of YY1. The frequent alteration in the YY1-RKIP balance might represent a marker of malignant progression and be exploited for therapeutic interventions in HCC.