Anil K. Tyagi

Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells

Silymarin, a defined mixture of natural flavonoid, has recently been shown to have potent cancer chemopreventive efficacy against colon carcinogenesis in rat model; however, the mechanism of such efficacy is not elucidated. Here, using pure active agent in silymarin, namely silibinin, we show its antiproliferative and apoptotic effects, and associated molecular alterations in human colon carcinoma HT-29 cells. Silibinin treatment of cells at 50–100 μg/ml doses resulted in a moderate to very strong growth inhibition in a dose- and a time-dependent manner, which was largely due to a G0/G1 arrest in cell cycle progression; higher dose and longer treatment time also caused a G2/M arrest. In mechanistic studies related its effect on cell cycle progression, silibinin treatment resulted in an upregulation of Kip1/p27 and Cip1/p21 protein as well as mRNA levels, and decreased CDK2, CDK4, cyclin E and cyclin D1 protein levels together with an inhibition in CDK2 and CDK4 kinase activities. In other studies, we observed that G2/M arrest by silibinin was associated with a decrease in cdc25C, cdc2/p34 and cyclin B1 protein levels, as well as cdc2/p34 kinase activity. In the studies assessing biological fate of silibinin-treated cells, silibinin-induced cell cycle arrest and growth inhibition were not associated with cellular differentiation, but caused apoptotic death. The quantitative apoptosis analysis showed up to 15% apoptotic cell death after 48 h of silibinin treatment. Interestingly, silibinin-induced apoptosis in HT-29 cells was independent of caspases activation, as all caspases inhibitor did not reverse silibinin-induced apoptosis. This observation was further confirmed by the findings showing a lack in caspases activity increase and caspases and PARP cleavage as well as a lack in cytochrome c release in cytosol following silibinin treatment of HT-29 cells. Additional studies conducted in mice showed that silibinin doses found effective in HT-29 cells are achievable in plasma, which increases the significance of the present findings and their possible translation in in vivo anticancer efficacy of silibinin against colon cancer. Together, these results identify molecular mechanisms of silibinin efficacy as a cell cycle regulator and apoptosis inducer in human colon carcinoma HT-29 cells, and justify further studies to investigate potential usefulness of this nontoxic agent in colon cancer prevention and intervention.

Grape seed extract inhibits advanced human prostate tumor growth and angiogenesis and upregulates insulin-like growth factor binding protein-3

Dietary intake of many fruits and vegetables has been shown to be associated with reduced risk of cancer. We investigated the in vivo efficacy of grape seed extract (GSE, patented as Traconol) against prostate cancer (PCA) and associated molecular events. Athymic nude mice were implanted with hormone‐refractory human prostate carcinoma DU145 cells and fed with 100 and 200 mg/kg/day (5 days/week) doses of GSE for 7 weeks. At the end of experiment, tumors were immunohistochemically analyzed for cell proliferation, apoptosis and angiogenesis. Our data show that GSE feeding strongly inhibited tumor growth that accounted for 59–73% (p < 0.001) inhibition in tumor volume and 37–47% (p < 0.05) decrease in tumor weight at the end of the experiment. It did not show any significant change in body weight gain profile and diet consumption. Immunohistochemical analysis of tumors showed that GSE decreases proliferation index by 51–66% (p < 0.001) and increases apoptotic index by 3–4‐fold (p < 0.001). CD31 staining for endothelial cells, showed decrease in intratumoral microvasculature in GSE‐fed group of mice. Control tumors showed 64.0 ± 1.6 CD31 positive cells/400× field compared to 23.2 ± 0.9 and 15.7 ± 0.08 (p < 0.001) CD31 positive cells in 100 and 200 mg/kg doses of GSE‐treated tumors, respectively. GSE strongly inhibited (47–70%, p < 0.05) vascular endothelial growth factor (VEGF) secretion in conditioned medium by DU145 cells. Recently, the circulating level of insulin‐like growth factor binding protein (IGFBP)‐3 is shown to inversely related with PCA risk, growth and prognosis. Consistent with this, we observed 6–7‐fold (p < 0.001) increase in tumor‐secreted levels of IGFBP‐3 after GSE feeding. In other immunohistochemical studies, compared to controls, tumor xenografts from GSE‐fed groups of mice showed a moderate decrease in VEGF but an increase in IGFBP‐3 levels. These findings suggest that GSE possesses in vivo anticancer efficacy against hormone‐refractory human PCA, which is associated with its antiproliferative, proapoptotic and antiangiogenic activities together with upregulation of IGFBP‐3.

IgG particle formation during filling pump operation: A case study of heterogeneous nucleation on stainless steel nanoparticles

This study investigated factors associated with vial filling with a positive displacement piston pump leading to formation of protein particles in a formulation of an IgG. We hypothesized that nanoparticles shed from the pumps solution-contact surfaces nucleated protein aggregation and particle formation. Vials of IgG formulation filled at a clinical manufacturing site contained a few visible particles and about 100,000 particles (1.5-3 microm) per mL. In laboratory studies with the same model (National Instruments FUS-10) of pump, pumping of 20 mg/mL IgG formulation resulted in about 300,000 particles (1.5-3 microm) per mL. Pumping of protein-free formulation resulted in 13,000 particles (1.5-15 microm) per mL. More than 99% of the particles were 0.25-0.95 microm in size. Mixing of protein-free pumped solution with an equal volume of 40 mg/mL IgG resulted in 300,000 particles (1.5-15 microm) per mL. Also, mixing IgG formulation with 30,000/mL stainless steel nanoparticles resulted in formation of 30,000 protein microparticles (1.5-15 microm) per mL. Infrared spectroscopy showed that secondary structure of IgG in microparticles formed by pumping or mixing with steel nanoparticles was minimally perturbed. Our results document that nanoparticles of foreign materials shed by pumps can serve as heterogeneous nuclei for formation of protein microparticles.

Oral Silibinin Inhibits Lung Tumor Growth in Athymic Nude Mice and Forms a Novel Chemocombination with Doxorubicin Targeting Nuclear Factor κB–Mediated Inducible Chemoresistance

The acute and cumulative dose-related toxicity and drug resistance, mediated via nuclear factor κB (NFκB), of anthracycline anticancer drugs pose a major problem in cancer chemotherapy. Here, we report that oral silibinin (a flavanone) suppresses human non–small-cell lung carcinoma A549 xenograft growth (P = 0.003) and enhances the therapeutic response (P < 0.05) of doxorubicin in athymic BALB/c nu/nu mice together with a strong prevention of doxorubicin-caused adverse health effects. Immunohistochemical analyses of tumors showed that silibinin and doxorubicin decrease (P < 0.001) proliferation index and vasculature and increase (P < 0.001) apoptosis; these effects were further enhanced (P < 0.001) in combination treatment. Pharmacologic dose of silibinin (60 μmol/L) achieved in animal study was biologically effective (P < 0.01 to 0.001, growth inhibition and apoptosis) in vitro in A549 cell culture together with an increased efficacy (P < 0.05 to 0.001) in doxorubicin (25 nmol/L) combination. Furthermore, doxorubicin increased NFκB DNA binding activity as one of the possible mechanisms for chemoresistance in A549 cells, which was inhibited by silibinin in combination treatment. Consistent with this, silibinin inhibited doxorubicin-caused increased translocation of p65 and p50 from cytosol to nucleus. Silibinin also inhibited cyclooxygenase-2, an NFκB target, in doxorubicin combination. These findings suggest that silibinin inhibits in vivo lung tumor growth and reduces systemic toxicity of doxorubicin with an enhanced therapeutic efficacy most likely via an inhibition of doxorubicin-induced chemoresistance involving NFκB signaling.

Synergistic Anti-Cancer Effects of Grape Seed Extract and Conventional Cytotoxic Agent Doxorubicin Against Human Breast Carcinoma Cells

With an approach to enhance the efficacy of chemotherapy agents against breast cancer treatment, here, we investigated the anti-cancer effects of grape seed extract (GSE) and doxorubicin (Dox), either alone or in combination, in estrogen receptor-positive MCF-7 and receptor-negative MDA-MB468 human breast carcinoma cells. GSE (25–200 µg/ml) treatment of cells resulted in 16–72% growth inhibition and 9–33% cell death, in a dose- and a time-dependent manner. In other studies, Dox (10–100 nM) treatment showed 23–96% growth inhibition and 10–55% cell death. Based on these results, several combinations of GSE (25–100 µg/ml) with Dox (10–75 nM) were next assessed for their synergistic, additive and/or antagonistic efficacy towards cell growth inhibition and death. In both MCF-7 and MDA-MB468 cells, a combination of 100 µg/ml GSE with 25–75 nM Dox treatment for 48 h showed a strong synergistic effect [combination index (CI) < 0.5] in cell growth inhibition, but mostly an additive effect (CI ∼ 1) in cell death. In cell-cycle progression studies, GSE plus Dox combination resulted in a moderate increase in G1 arrest in MCF-7 cells compared to each agent alone. GSE plus Dox combination showed a very strong and significant G1 arrest in MDA-MB468 cells when compared with Dox alone, however, it was less than that observed with GSE alone. In quantitative apoptosis studies, GSE and Dox alone and in combination showed comparable apoptotic death of MCF-7 cells, however, a combination of the two was inhibitory to Dox induced apoptosis in MDA-MB468 cells. This was further confirmed in another estrogen receptor-negative MDA-MB231 cell line, in which GSE and Dox combination strongly inhibited cell growth but did not show any increase in apoptotic cell death caused by Dox. Together, these results suggest a strong possibility of synergistic efficacy of GSE and Dox combination for breast cancer treatment, independent of estrogen receptor status of the cancer cell.

Chemopreventive effects of silymarin and silibinin on N-butyl-N-(4-hydroxybutyl) nitrosamine induced urinary bladder carcinogenesis in male ICR mice.

Effective strategies are lacking for the management of urinary bladder cancer for which smoking is a potential risk factor. Herein, we evaluated chemoprevention of urinary bladder cancer by natural chemopreventive agents, silymarin and silibinin, in a preclinical animal (ICR mouse) model of bladder cancer induced by tobacco smoke carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN). Mice were fed p.o. with saline or OH-BBN (0.05%, w/v) in drinking water for 6 weeks or with silymarin or silibinin (200 mg/kg body weight for both) starting 1 week before OH-BBN exposure for 51 weeks. Silymarin and silibinin strongly arrested OH-BBN–induced tumor progression at the stage of mucosal dysplasia with a striking reduction in papillary nodular dysplasia as well as invasive carcinoma. Some silymarin- or silibinin-treated mice developed no urothelial lesions in spite of OH-BBN exposure. Immunohistochemical analyses at study conclusion revealed that silymarin and silibinin decreased cell proliferation by 42% (P < 0.001) and 44% (P < 0.001) and increased apoptosis by 4-fold (P < 0.05) and 6-fold (P < 0.05) in OH-BBN–induced urothelium, respectively. Antiproliferative and apoptotic effects of silymarin and silibinin were associated with decreases in (a) cyclin D1 protein level and extracellular signal–regulated kinase-1/2 phosphorylation and in (b) protein levels of survivin and nuclear phospho-p65 (Ser276 and Ser536), respectively. Together, these results suggest that silymarin and silibinin inhibit chemically induced urinary bladder tumor growth and progression possibly by inhibiting cell proliferation and enhancing apoptosis. [Mol Cancer Ther 2007;6(12):3248–55]

Development of a freeze-stable formulation for vaccines containing aluminum salt adjuvants.

Vaccines containing aluminum salt adjuvants are prone to inactivation following exposure to freeze-thaw stress. Many are also prone to inactivation by heat. Thus, for maximum potency, these vaccines must be maintained at temperatures between 2 degrees C and 8 degrees C which requires the use of the cold chain. Nevertheless, the cold chain is not infallible. Vaccines are subject to freezing during both transport and storage, and frozen vaccines are discarded (under the best circumstances) or inadvertently administered despite potentially reduced potency. Here we describe an approach to minimize our reliance on the proper implementation of the cold chain to protect vaccines from freeze-thaw inactivation. By including PEG 300, propylene glycol, or glycerol in a hepatitis B vaccine, particle agglomeration, changes in the fluorescence emission spectrum--indicative of antigen tertiary structural changes--and losses of in vitro and in vivo indicators of potency were prevented following multiple exposures to -20 degrees C. The effect of propylene glycol was examined in more detail and revealed that even at concentrations too low to prevent freezing at -10 degrees C, -20 degrees C, and -80 degrees C, damage to the vaccine could be prevented. A pilot study using two commercially available diphtheria, tetanus toxoid, and acellular pertussis (DTaP) vaccines suggested that the same stabilizers might protect these vaccines from freeze-thaw agglomeration as well. It remains to be determined if preventing agglomeration of DTaP vaccines preserves their antigenic activity following freeze-thaw events.

Characterization of the freeze sensitivity of a hepatitis B vaccine

Recent studies have revealed that vaccines containing aluminum adjuvant are exposed to sub-zero temperatures while in the cold chain more frequently than was previously believed. This raises concerns that these freeze-sensitive vaccines may be damaged and offer inadequate protection. This study was undertaken to characterize the immediate qualitative changes of one such vaccine, hepatitis B, caused by freeze exposure. Hepatitis B vaccine was subjected to freezing temperatures ranging from 0°C to -20°C for up to three episodes with durations ranging from 1 hour to 7 days. The vaccine was analyzed for freezing point, particle size distribution, tertiary structure, and in vitro and in vivo potency. Whether or not hepatitis B vaccine freezes was shown to be dependent on an array of factors including temperature, rate of temperature change, duration of exposure, supercooling effects, and vibration. Vaccine exposed to “mild” freezing (-4°C or warmer) temperatures did not freeze and remained qualitatively unaltered. Single or repeated freezing events at temperatures of -10°C or lower were associated with aggregation of the adjuvant-antigen particles, structural damage of the antigen, and reduction of immunogenicity in mice. Damage to the vaccine increased with duration of freezing, lower temperature, and the number of freezing episodes. With vibration, vaccine froze at -6°C after 1 hour and damage occurred. Freezing and freeze damage to vaccines containing aluminum adjuvant represents a real risk to effectiveness of immunizations and should be prevented by strengthening the cold chain system or, alternatively, development of freeze-stable vaccine formulations.

Characterization of a thermostable hepatitis B vaccine formulation.

Cold chain requirements for vaccine storage and distribution are both economic and logistical burdens for immunization programs, especially those in lower-resource settings. Inadvertent exposure of vaccines to both heat and freezing temperatures within such cold chains are frequently occurring problems in both developing and industrialized countries. Here we report on a new hepatitis B vaccine formulation that is stable against repeated freezing at -20 degrees C and is also stable for 12 months at 37 degrees C. The thermostable vaccine contains all the components of the original vaccine plus 7.5% (v/v) propylene glycol, 40mM phosphate, and 40mM histidine with a final pH of 5.2. The propylene glycol is responsible for the freeze stability while the other components are essential for the heat stability. This formulation was found to be well tolerated in rabbits without any significant local or systemic side effects. The improved stability of this hepatitis B vaccine could be a key factor in ensuring vaccine effectiveness, extending immunization coverage, simplifying immunization logistics, and reducing the costs associated with the cold chain.

Interaction of l-alanosine (nsc 153,353) with enzymes metabolizing l-aspartic acid, l-glutamic acid and their amides.

Abstract A comprehensive analysis has been made of the manner in which l -alanosine [ l -2-amino-3-( N -nitroso, N -hydroxy) aminopropionic acid] interacts with the enzymes responsible for the metabolism of the dicarboxylic amino acids and their amides. The drug impedes the transport of l -aspartic acid and, to a lesser degree, of l -glutamic acid, l -asparagine and l -glutamine by lymphoblasts. in vitro; in each of these instances, inhibition is apparently competitive in type. Of the enzymes involved in the metabolism of l -aspartic acid, adenylosuccinate synthetase (EC 6.3.4.4). SAICAR synthetase (5-amino-4-imidazole- N -succinocarboxamide ribonucleotide synthetase) (EC 6.3.2.6). l -aspartyl tRNA synthetase (EC 6.1.1.12), l -aspartate transcarbamylase (EC 2.1.3.2) and l -aspartate aminotransferase (EC 2.6.1.1) were inhibited by l -alanosine; moreover, each of these enzymes except l -aspartyl tRNA synthetase accepted the antibiotic as substrate, although at substantially diminished rates. Of the enzymes involved in the metabolism of l -glutamic acid, l -alanosine inhibited only l -glutamine synthetase (EC 6.3.1.2) and l -glutamate decarboxylase (EC 4.1.1.15) to a prominent degree; this last enzyme was also capable of decarboxylating l -alanosine. Of the enzymes metabolizing l -asparagine and/or l -glutamine. only the l -glutaminase activity of l -asparagine amidohydrolase (EC 3.5.1.1) (with l -glutamine as substrate) and, to a lesser degree, carbamyl phosphate synthetase II (EC 2.7.2.9) were inhibited by the antibiotic. Although l -alanosine provokes a rise in the concentration of inosinic acid (IMP) in vitro, pointing to the conclusion that the drug is capable of inhibiting adenylosuccinate synthetase under these circumstances, no such rise was seen in vivo either in tumor or liver. However, 1 and 5 hr after administration l -alanosine depressed hepatic ATP and NAD pools, an effect which indicates that the drug is. in fact, restricting the intracellular concentration of adenine nucleotides. Of the metabolites of l -alanosine formed in vitro, α-decarboxy alanosine, α-keto alanosine, a-hydroxy alanosine. alanosyl IMP and N -carbamyl l -alanosine did not inhibit adenylosuccinate synthetase to any prominent degree, whereas the metabolite generated by SAICAR synthetase powerfully inhibited this enzyme, with a K i , of 0.3μM. Parenteral therapeutic doses of l -alanosine produced striking increases in the concentrations of l -aspartic acid in tumor and liver as well as of l -aspartic and l -glutamic acids in urine. It is concluded that the N -hydioxy, N -nitroso functionality of l -alanosine is analogous in structure to the β-carboxyls of l -aspartic and l -glutamic acids, respectively; this analogy permits l -alanosine to be anabolized and catabolized via several of the enzymatic routes which ordinarily operate on these dicarboxylic amino acids.