Melanoma Cell Reprogramming and Awakening of Antitumor Immunity as a Fingerprint of Hyper-Harmonized Hydroxylated Fullerene Water Complex (3HFWC) and Hyperpolarized Light Application In Vivo.

In our recent study, we showed that in vitro treatment of melanoma cells with hyperpolarized light (HPL) as well as with the second derivative of fullerene, hyper-harmonized hydroxylated fullerene water complex (3HFWC) reduced viability of cells by decreasing their proliferative capacity and inducing senescence and reprogramming towards a normal, melanocytic phenotype. Therefore, we wanted to determine whether these effects persisted in vivo in the syngeneic mouse melanoma model with a combined treatment of HPL irradiation and 3HFWC per os. Our results demonstrated the potent antitumor effects of 3HFWC nanosubstance assisted by HPL irradiation. These effects were primarily driven by the stimulation of melanoma cell growth arrest, the establishment of a senescent phenotype, and melanocytic differentiation on the one hand, and the awakening of the antitumor immune response on the other. In addition, the combined treatment reduced the protumorigenic activity of immune cells by depleting T regulatory cells, myeloid-derived suppressors, and M2 macrophages. The support of the 3HFWC substance by HPL irradiation may be the axis of the new approach design based on tumor cell reprogramming synchronized with the mobilization of the host's protective immune response.

Carborane Analogues of Fenoprofen Exhibit Improved Antitumor Activity.

Fenoprofen is a widely used nonsteroidal anti-inflammatory drug (NSAID) against rheumatoid arthritis, degenerative joint disease, ankylosing spondylitis and gout. Like other NSAIDs, fenoprofen inhibits the synthesis of prostaglandins by blocking both cyclooxygenase (COX) isoforms, COX-1 the "house-keeping" enzyme and COX-2 the induced isoform from pathological stimuli. Unselective inhibition of both COX isoforms results in many side effects, but off-target effects have also been reported. The steric modifications of the drugs could afford the desired COX-2 selectivity. Furthermore, NSAIDs have shown promising cytotoxic properties. The structural modification of fenoprofen using bulky dicarba-closo-dodecaborane(12) (carborane) clusters and the biological evaluation of the carborane analogues for COX inhibition and antitumor potential showed that the carborane analogues exhibit stronger antitumor potential compared to their respective aryl-based compounds.

GSTA4 Governs Melanoma Immune Resistance and Metastasis.

IMPLICATIONS: Considering the importance of GSTA4 in controlling IFNγ responsiveness and the metastatic potential of other melanoma cells, our results highlight a novel mechanism whereby cancer cells escape from host immunity and gain metastatic ability by acquiring resistance to oxidative stress responses through the upregulation of GSTA4.

Effect of chain length on the cytotoxic activity of (alkyl-ω-ol)triphenyltin(IV) loaded into SBA-15 nanostructured silica and in vivo study of SBA-15~Cl|Ph3Sn(CH2)8OH.

A series of nanostructured SBA-15-based materials functionalized with the tetraorganotin(IV) metallodrugs Ph3Sn(CH2)nOH (n = 3, 4, 6, 8 and 11) are synthesized and structurally characterized by different techniques used in solid-state chemistry. The cytotoxicity of both the organotin(IV) compounds and the tin-functionalized SBA-15 materials are studied against different cancer cell lines observing that the materials have similar cytotoxic activity in comparison with the free organotin compounds in terms of mass. However, considering that the percentage of active metal compound loaded into material is low, the utilization of mesoporous silica as drug vehicle clearly improves the cytotoxic effectiveness of metal-based drugs against cancer cells. One of the most potent between all tested systems is material SBA-15~Cl|Ph3Sn(CH2)8OH. Its cytotoxicity seems to come from additional mechanisms apart from apoptosis provoking cell reprogram in B16 melanoma into more mature and less aggressive phenotype. Moderated production of ROS/RNS is probably in the background of observed phenomenon. Obtained results are further confirmed in syngeneic mouse model of melanoma in C57BL6 mice. The in vivo results show that SBA-15 do not disturb tumor growth, while both Ph3Sn(CH2)8OH and SBA-15~Cl|Ph3Sn(CH2)8OH significantly decreases tumor volume with an enhancement of the antitumor potential of the tetraorganotin(IV) compound upon immobilization in SBA-15.

Carboranyl Analogues of Mefenamic Acid and Their Biological Evaluation.

Mefenamic acid represents a widely used nonsteroidal anti-inflammatory drug (NSAID) to treat the pain of postoperative surgery and heavy menstrual bleeding. Like other NSAIDs, mefenamic acid inhibits the synthesis of prostaglandins by nonselectively blocking cyclooxygenase (COX) isoforms COX-1 and COX-2. For the improved selectivity of the drug and, therefore, reduced related side effects, the carborane analogues of mefenamic acid were evaluated. The ortho-, meta-, and para-carborane derivatives were synthesized in three steps: halogenation of the respective cluster, followed by a Pd-catalyzed B-N coupling and hydrolysis of the nitrile derivatives under acidic conditions. The COX inhibitory activity and cytotoxicity for different cancer cell lines revealed that the carborane analogues have stronger antitumor potential compared to their parent organic compound.

NKG2D defines tumor-reacting effector CD8+ T cells within tumor microenvironment.

For successful immunotherapy for cancer, it is important to understand the immunological status of tumor antigen-specific CD8+ T cells in the tumor microenvironment during tumor progression. In this study, we monitored the behavior of B16OVA-Luc cells in mice immunized with a model tumor antigen ovalbumin (OVA). Using bioluminescence imaging, we identified the time series of OVA-specific CD8+ T-cell responses during tumor progression: initial progression, immune control, and the escape phase. As a result of analyzing the status of tumor antigen-specific CD8+ cells in those 3 different phases, we found that the expression of NKG2D defines tumor-reacting effector CD8+ T cells. NKG2D may control the fate and TOX expression of tumor-reacting CD8+ T cells, considering that NKG2D blockade in OVA-vaccinated mice delayed the growth of the B16OVA-Luc2 tumor and increased the presence of tumor-infiltrating OVA-specific CD8+ T cells.

Development of genistein-loaded gold nanoparticles and their antitumor potential against prostate cancer cell lines.

Soy isoflavone genistein (Gen) exerts beneficial effects against prostate cancer cells in vitro and in vivo. However, its use as a chemoprevention/therapeutic agent is largely limited due to its low bioavailability. In this study we synthesized two variants of a new delivery system, genistein-gold nanoparticles conjugates Gen@AuNPs1 and Gen@AuNPs2, by an environmentally friendly method, using a dual role of Gen to reduce Au3+ and stabilize the formed AuNPs, with no additional component. The formation of Gen@AuNPs was confirmed via UV-Vis spectroscopy, FTIR, and Raman spectra measurements. The spherical shape and uniform size of Gen@AuNPs1 and Gen@AuNPs2 (10 ± 2 and 23 ± 3 nm, respectively), were determined by transmission electron microscopy. The nano-conjugates also varied in hydrodynamic diameter (65.0 ± 1.7 and 153.0 ± 2.2 nm) but had similar negative zeta potential (-35.0 ± 2.5 and -37.0 ± 1.6 mV), as measured by dynamic light scattering. The Gen loading was estimated to be 46 and 48%, for Gen@AuNPs1 and Gen@AuNPs2, respectively. The antiproliferative activities of GenAuNPs were confirmed by MTT test in vitro on three malignant prostate carcinoma cell lines (PC3, DU 145, and LNCaP), while selectivity toward malignant phenotype was confirmed using non-cancerous MRC-5 cells. Flow cytometric analysis showed that the inhibition on cell proliferation of more potent Gen@AuNPs1 nano-conjugate is comparable with the effects of free Gen. In conclusion, the obtained results, including physicochemical characterization of newly synthesized AuNPs loaded with Gen, cytotoxicity, and IC50 assessments, indicate their stability and bioactivity as an antioxidant and anti-prostate cancer agent, with low toxicity against human primary cells.

Daidzein upregulates anti-aging protein Klotho and NaPi 2a cotransporter in a rat model of the andropause.

In a rat model of the andropause we aimed to examine the influence of daidzein, soy isoflavone, on the structure and function of parathyroid glands (PTG) and the expression levels of some of the crucial regulators of Ca2+ and Pi homeostasis in the kidney, and to compare these effects with the effects of estradiol, serving as a positive control. Middle-aged (16-month-old) male Wistar rats were divided into the following groups: sham-operated (SO), orchidectomized (Orx), orchidectomized and estradiol-treated (Orx+E; 0.625mg/kg b.w./day, s.c.) as well as orchidectomized and daidzein-treated (Orx+D; 30mg/kg b.w./day, s.c.) group. Every treated group had a corresponding control group. PTH serum concentration was decreased in Orx+E and Orx+D groups by 10% and 21% (p<0.05) respectively, in comparison with the Orx. PTG volume was decreased in Orx+E group by 16% (p<0.05), when compared to the Orx. In Orx+E group expression of NaPi 2a was lower (p<0.05), while NaPi 2a abundance in Orx+D animals was increased (p<0.05), when compared to Orx. Expression of PTH1R was increased (p<0.05) in Orx+E group, while in Orx+D animals the same parameter was decreased (p<0.05), in comparison with Orx. Klotho expression was elevated (p<0.05) in Orx+D rats, in regard to Orx. Orx+D induced reduction in Ca2+/creatinine and Pi/creatinine ratio in urine by 32% and 16% (p<0.05) respectively, in comparison with Orx. In conclusion, presented results indicate the more coherent beneficial effects of daidzein compared to estradiol, on disturbed Ca2+ and Pi homeostasis, and presumably on bone health, in the aging male rats.

The Dark Side of IFN-γ: Its Role in Promoting Cancer Immunoevasion.

Interferon-γ (IFN-γ) is a pleiotropic cytokine that has long been praised as an important effector molecule of anti-tumor immunity, capable of suppressing tumor growth through various mechanisms. On the contrary to such a bright side of IFN-γ, it has also been involved in promoting an outgrowth of tumor cells with immunoevasive phenotype suggesting an existence of a dark "tumor-promoting" side effect of IFN-γ. In this review, we will summarize this multi-functional role of IFN-γ in tumor context, how it promotes changes in tumor phenotype towards increased fitness for growth in immunocompetent host. Furthermore, we summarize how IFN-γ is involved in homeostatic or cancer-triggered mechanisms to establish an immunosuppressive tumor microenvironment.

Alpha-1-Antitrypsin Antagonizes Cisplatin-Induced Cytotoxicity in Prostate Cancer (PC3) and Melanoma Cancer (A375) Cell Lines.

Increased circulating alpha-1-antitrypsin (AAT) correlates with cancer stage/aggressiveness, but its role in cancer biology is unclear. We revealed antagonistic effect of AAT to cisplatin-induced cytotoxicity in prostate (PC3) and melanoma (A375) cancer cell lines. Moreover, AAT abrogated cytotoxicity of MEK inhibitor U0126 in PC3 cell line. Weaker antagonistic effect of AAT on cytotoxicity of PI3/Akt and NF-kB inhibitors was also observed. In addition, cisplatin increased AAT gene expression in transfected PC3 cells. However, AAT derived from transfected PC3 cells did not antagonize cisplatin-induced cytotoxicity. In conclusion, these results suggest possible association between high circulating AAT and cisplatin resistance.

Evaluation of functionalized mesoporous silica SBA-15 as a carrier system for Ph3Sn(CH2)3OH against the A2780 ovarian carcinoma cell line.

SBA-15|Sn3, a mesoporous silica-based material (derivative of SBA-15) loaded with an organotin compound Ph3Sn(CH2)3OH (Sn3), possesses improved antitumor potential against the A2780 high-grade serous ovarian carcinoma cell line in comparison to Sn3. It is demonstrated that both the compound and the nanostructured material are internalized by the A2780 cells. A similar mode of action of Sn3 and SBA-15|Sn3 against the A2780 cell line was found. Explicitly, induction of apoptosis, caspase 2, 3, 8 and 9 activation, accumulation of cells in the hypodiploid phase as well as accumulation of ROS were observed. Interestingly, Sn3 loaded in the mesoporous silica-based material needed to reach a concentration 3.5 times lower than the IC50 value of the Sn3 compound, pointing out a higher effect of the SBA-15|Sn3 than Sn3 alone. Clonogenic potential, growth in 3D culture as well as mobility of cells were disturbed in the presence of SBA-15|Sn3. Such behavior could be associated with the suppression of p-38 MAPK. Less profound effect of Sn3 compared to SBA-15|Sn3 could be attributed to a different regulation of p-38 and STAT-3, which are mainly responsible for an appropriate cellular response to diverse stimuli or metastatic properties.

Anticancer Activity of Organogallium(III) Complexes in Colon Cancer Cells.

In vitro antitumor activity of various organogallium(III) complexes (1-8) has been tested against CT26CL25, HCT116, SW480 colon cancer cell lines. CV and MTT assays were used to assess on the antiproliferative effect of investigated organogallium(III) complexes. From the investigated complexes, the most active was found to be tetranuclear compound 8 against CT26CL25 cells. Flow cytometric analysis of the CT26CL25 cells upon the treatment with 8 was performed in order to determine the role of apoptosis, caspase activation, autophagy and proliferation rate on the cell death caused with this compound. Results indicate cytotoxic potential of the tetranuclear complex 8 by inducing caspase independent apoptosis and blocking most of the cells before first division.

Biological Potential of Halfsandwich Ruthenium(II) and Iridium (III) Complexes.

In vitro studies with the ruthenium(II) and analogous iridium(III) complexes [Ru(η6- p-cymene)Cl2{Ph2PCH2CH2CH2S(O)xPh-κP}], [Ru(η6-p-cymene)Cl{Ph2PCH2CH2CH2S(O)xPh- κP,κS}][PF6] (1-4), [Ir(η5-C5Me5)Cl2{Ph2PCH2CH2CH2S(O)xPh-κP}] and [Ir(η5-C5Me5)Cl{Ph2 PCH2CH2CH2S(O)xPh-κP,κS}][PF6] (5-8; x = 0, 1) revealed the high selectivity toward the 8505C, A253, MCF-7, SW480 and 518A2 cancer cell lines. Thus, the cationic ruthenium complex 4 proved to be the most selective one. In case of the neutral and cationic ruthenium complexes 1-4 the caspase-dependent apoptotic cell death was proven as the main cause of the drug's tumoricidal action on 8505C cell line.

The NO-modified HIV protease inhibitor as a valuable drug for hematological malignancies: Role of p70S6K.

Covalent attachment of NO to the first approved HIV protease inhibitor Saquinavir (Saq-NO) expands the therapeutic potential of the original drug. Apart from retained antiviral activity, the modified drug exerts strong antitumor effects and lower toxicity. In the present study, we have evaluated the sensitivity of different hematological malignancies to Saq-NO. Saq-NO efficiently diminished the viability of Jurkat, Raji, HL-60 and K562 cells. While Jurkat and Raji cells (established from pediatric patients) displayed abrogated proliferative potential, HL-60 and K652 cells (originated from adults) exposed to Saq-NO treatment underwent caspase dependent apoptosis. In addition, similar sensitivity to Saq-NO was observed in mononuclear blood cells obtained from pediatric patients with acute lymphoblastic leukemia (ALL) and adult patients with acute myeloid leukemia (AML). Western blot analysis indicated p70S6 kinase as a possible intracellular target of Saq-NO action. Moreover, the addition of a NO moiety to Lopinavir resulted in improved antitumor potential as compared to the parental compound, suggesting that NO-derived HIV protease inhibitors are a potential new source of anticancer drugs with unique mode of action.

Improved in vitro antitumor potential of (O,O'-Diisobutyl-ethylenediamine-N,N'-di-3-propionate)tetrachloridoplatinum(IV) complex under normoxic and hypoxic conditions.

(O,O'-Diisobutyl-ethylenediamine-N,N'-di-3-propionate)tetrachloridoplatinum(IV), [PtCl4(iBu2eddp)], shows an improved pharmacological profile in comparison to cisplatin. This is manifested through accelerated dying process led by necrotic cell death, reflected through mitochondrial collapse, strong ATP depletion and reactive oxygen species production. Loss of mitochondrial potential was further followed with intensive apoptosis that finalized with DNA fragmentation. Different dynamic of tumoricidal action could be partly ascribed to less affected repair mechanisms in comparison to cisplatin. Importantly, [PtCl4(iBu2eddp)] did not induce necrosis in primary fibroblasts suggesting different intracellular response of normal vs. tumor cells. This selectivity toward malignant phenotype is further confirmed by retained tumoricidal potential in hypoxic conditions, while cisplatin became completely inefficient.

Membrane steroid receptor-mediated action of soy isoflavones: tip of the iceberg.

Soy isoflavone's (genistein and daidzein in particular) biological significance has been thoroughly studied for decades, so we started from the premise that refreshed investigation approach in this field should consider identification of their new molecular targets. In addition to recently described epigenetic aspects of polyphenole action, the cell membrane constituents-mediated effects of soy isoflavones are worthy of special attention. Accordingly, the expanding concept of membrane steroid receptors and rapid signaling from the cell surface may include the prominent role of these steroid-like compounds. It was observed that daidzein strongly interacts with membrane estrogen receptors in adrenal medullary cells. At low doses, daidzein was found to stimulate catecholamine synthesis through extracellular signal-regulated kinase 1/2 or protein kinase A pathways, but at high doses, it inhibited catecholamine synthesis and secretion induced by acetylcholine. Keeping in mind that catecholamine excess can contribute to the cardiovascular pathologies and that catecholamine lack may lead to depression, daidzein application promises to have a wide range of therapeutic effects. On the other hand, it was shown in vitro that genistein inhibits LNCaP prostate cancer cells invasiveness by decreasing the membrane fluidity along with immobilization of the androgen receptor containing membrane lipid rafts, with down regulation of the androgen receptors and Akt signaling. These data are promising in development of the molecular pharmacotherapy pertinent to balanced soy isoflavone treatment of cardiovascular, psychiatric, and steroid-related malignant diseases.

Extracellular iron diminishes anticancer effects of vitamin C: an in vitro study.

In vitro studies have shown that hydrogen peroxide (H2O2) produced by high-concentration ascorbate and cell culture medium iron efficiently kills cancer cells. This provided the rationale for clinical trials of high-dose intravenous ascorbate-based treatment for cancer. A drawback in all the in vitro studies was their failure to take into account the in vivo concentration of iron to supplement cell culture media which are characterized by low iron content. Here we showed, using two prostate cancer cell lines (LNCaP and PC-3) and primary astrocytes, that the anticancer/cytotoxic effects of ascorbate are completely abolished by iron at physiological concentrations in cell culture medium and human plasma. A detailed examination of mechanisms showed that iron at physiological concentrations promotes both production and decomposition of H2O2. The latter is mediated by Fenton reaction and prevents H2O2 accumulation. The hydroxyl radical, which is produced in the Fenton reaction, is buffered by extracellular proteins, and could not affect intracellular targets like H2O2. These findings show that anticancer effects of ascorbate have been significantly overestimated in previous in vitro studies, and that common cell culture media might be unsuitable for redox research.

Organotin(IV)-loaded mesoporous silica as a biocompatible strategy in cancer treatment.

The strong therapeutic potential of an organotin(IV) compound loaded in nanostructured silica (SBA-15pSn) is demonstrated: B16 melanoma tumor growth in syngeneic C57BL/6 mice is almost completely abolished. In contrast to apoptosis as the basic mechanism of the anticancer action of numerous chemotherapeutics, the important advantage of this SBA-15pSn mesoporous material is the induction of cell differentiation, an effect unknown for metal-based drugs and nanomaterials alone. This non-aggressive mode of drug action is highly efficient against cancer cells but is in the concentration range used nontoxic for normal tissue. JNK (Jun-amino-terminal kinase)-independent apoptosis accompanied by the development of the melanocyte-like nonproliferative phenotype of survived cells indicates the extraordinary potential of SBA-15pSn to suppress tumor growth without undesirable compensatory proliferation of malignant cells in response to neighboring cell death.

Undecylprodigiosin conjugated monodisperse gold nanoparticles efficiently cause apoptosis in colon cancer cells in vitro.

Bacterial pigment undecylprodigiosin (UP) was produced using Streptomyces sp. JS520 and conjugated to monodisperse gold nanoparticles (UP-Au). Both UP and UP-Au showed cytocidal activity towards melanoma (A375), lung carcinoma (A549), breast cancer (MCF-7) and colon cancer (HCT-116) cells, inducing apoptosis with IC50 values ranging from 0.4 to 4 µg ml-1. Unconjugated UP had a tendency to lose its activity over time and to change biophysical characteristics over pH. The loss of the pigment potency was overcome by conjugation with gold nanoparticles. UP-Au exhibited high stability over pH 3.8 to 7.4 and its activity remained unaffected in time. Nano-packing changed the mechanism of UP toxicity by converting the intracellular signals from a mitochondrial dependent to a mitochondrial independent apoptotic process. The availability of nonpyrogenic UP in high amounts, together with specific anticancer activity and improved stability in the complex with gold nanoparticles, presents a novel platform for further development of UP-Au complexes as an anticancer drug suitable for clinical applications.

A Key Role of Autophagy in Osteoblast Differentiation on Titanium-Based Dental Implants.

Autophagy plays an important role in embryogenesis, for the maintenance of tissue homeostasis and the elimination of damaged subcellular structures. Furthermore, autophagy could be a mode of physiological cell death and also be implicated in cell differentiation. Thus, we hypothesized that autophagy may have an impact on the differentiation of osteoblast cells influenced by various titanium-based surfaces. Interactions between smooth, commercially available pure titanium (Ti cp), rough Ticer, acid-etched Ti cp (SS) and M1-M3 (comprised of the monoclinic phase of sodium-titanium oxides and rutile; M2 contains amorphous calcium phosphates) and human osteoblast cells were investigated. Immunofluorescent staining was used for detecting autophagy, cell cluster formation and collagen type I (Col-1) expression. Flow cytometry was employed to identify autophagy, the production of endogenous nitric oxide (NO) and the size and granularity of the cells. Rough surfaces caused osteoblast differentiation via the autophagic-dependent PI3/Akt signalling pathway. These surfaces induced the formation of discrete populations of large, granular cells, i.e. mature osteoblasts. In addition, M1-M3 provoked the development of a third population of small, granular cells, responsible for cell cluster formation, which are important for the formation of bone noduli and mineralisation. The same surfaces induced faster osteoblast maturation and enhanced NO production, a hallmark of the already mentioned processes. Neither the mature osteoblasts nor the small cells appeared after the inhibition of autophagy. Inhibition of autophagy also prevented cell cluster formation. We demonstrate that autophagy plays an essential role in the osteoblast differentiation on titanium-based surfaces with rough topography.