Establishment of a human neonatal hepatocyte cell line.

Hepatocytes are routinely used to generate and identify drug metabolites and hepatic toxicity. Primary cultures of human hepatocytes are the model cell of choice for most of these pharmacological and toxicological studies. However, major problems are encountered with primary liver cell cultures: the dwindling availability of viable livers, hepatocytes having a limited life span, the loss of liver-specific functions in culture, and the donor to donor variability. These limitations have created a significant need for an in vitro hepatocyte system, which has both the potential for use in toxicological and pharmaceutical studies as well as clinical applications. Ectopic expression of human telomerase reverse transcriptase (hTERT) is one of the major strategies used to develop immortalized cells. Immortalization of primary cells using hTERT allows retention of the original cellular characteristics and functions and avoids some of the genetic and phenotypic instabilities associated with using known oncogenes. In the present study, we developed a cell line from human neonatal hepatocytes by transduction with a recombinant retrovirus expressing the hTERT gene. Induction of stable expression of hTERT in the neonatal cells led to immortalization of these cells. The cell line was cultured continuously for more than 25 passages, equivalent to >25 population doublings, whereas the parental cells senesced within five passages. Analysis of telomerase activity as measured by telomeric repeat amplification protocol assay indicated elevated levels of telomerase activity in immortalized cells compared to the parental cells. These immortalized human hepatocytes cells maintained a normal diploid karyotype as well as the gene expression profile similar to that of human normal neonatal hepatocytes. The data suggest that these immortalized cells preserved some of the biological characteristics of hepatic progenitor cells and might be useful as an in vitro model for pharmacological and toxicity studies.

Green tea polyphenols and its constituent epigallocatechin gallate inhibits proliferation of human breast cancer cells in vitro and in vivo.

Tea [Camellia sinensis (Theaceae)] intake is second only to water in terms of worldwide popularity as a beverage. The Green tea polyphenols have been shown to have a protective effect in prostate cancer in various pre-clinical animal models and has been reported to be effective in several other cancer types as well. An inverse association between the risk of breast cancer and the intake of green tea has also been reported in Asian Americans. Several epidemiological studies have shown that breast cancer progression is delayed in the Asian population that consumes green tea on regular basis. In this study, we report the effectiveness of green tea polyphenols (GTP) and its constituent Epigallocatechin Gallate (EGCG) in tumor regression using both in-vitro cell culture models and in vivo athymic nude mice models of breast cancer. The anti-proliferative effect of GTP and EGCG on the growth of human breast cancer MDA-MB-231 cell was studied using a tetrazolium dye-based (MTT) assay. Both GTP and EGCG treatment had the ability to arrest the cell cycle at G1 phase as assessed by flow cytometry. The expression of Cyclin D, Cyclin E, CDK 4, CDK 1 and PCNA were down regulated over the time in GTP and EGCG treated experimental group, compared to the untreated control group as evaluated by western blot analysis for cell cycle proteins, which corroborated the G1 block. Nude mice inoculated with human breast cancer MDA-MB-231 cells and treated with GTP and EGCG were effective in delaying the tumor incidence as well as reducing the tumor burden when compared to the water fed and similarly handled control. GTP and EGCG treatment were also found to induce apoptosis and inhibit the proliferation when the tumor tissue sections were examined by immunohistochemistry. Our results suggest that GTP and EGCG treatment inhibits proliferation and induce apoptosis of MDA-MB-231 cells in-vitro and in-vivo. All together, these data sustain our contention that GTP and EGCG have anti-tumor properties.

Can homeopathic treatment slow prostate cancer growth?

BACKGROUND: Homeopathy is a complementary medicine widely used around the world. Despite extensive use of homeopathy for cancer and other serious conditions with reported success, clinical and laboratory research has been equivocal, and no rigorous research has been done on cancer. In 1999, the US National Cancer Institute evaluated the effects of homeopathic treatment of cancer from a clinic in India and has released a request for protocols to conduct further research into this treatment. Therefore, the authors conducted a series of carefully controlled laboratory studies evaluating the effects of commonly used homeopathic remedies in cell and animal models of prostate cancer. STUDY DESIGN: One hundred male Copenhagen rats were randomly assigned to either treatment or control groups after inoculation with prostate tumor cells. METHODS: Prostate tumor cells DU-145, LNCaP, and MAT-LyLu were exposed to 5 homeopathic remedies. Male Copenhagen rats were injected with MAT-LyLu cells and exposed to the same homeopathic remedies for 5 weeks. In vitro outcomes included tumor cell viability and apoptosis gene expression. In vivo outcomes included tumor incidence, volume, weight, total mortality, proliferating cell nuclear antigen (PCNA) expression, apoptotic cell death (terminal deoxynucleotidyl transferase mediated d-uridine triphosphate nick end labeling), and gene expression (rAPO-multiprobe). RESULTS: There were no effects on cell viability or gene expression in 3 prostate cell lines with any remedies at any exposure time. There was a 23% reduction in tumor incidence (P < .0001), and for animals with tumors, there was a 38% reduction in tumor volume in homeopathy-treated animals versus controls (P < .02). At time of killing, experimental animals with tumors had a 13% lower average tumor weight (P < .05). Tumors in these treated animals showed a 19% increase in apoptotic cell death (P < .05) and reduced PCNA-positive cells. CONCLUSIONS: The findings indicate that selected homeopathic remedies for the present study have no direct cellular anticancer effects but appear to significantly slow the progression of cancer and reduce cancer incidence and mortality in Copenhagen rats injected with MAT-LyLu prostate cancer cells.

Effect of homeopathic treatment on gene expression in Copenhagen rat tumor tissues.

BACKGROUND: Increasing evidence suggests that the inability to undergo apoptosis is an important factor in the development and progression of prostate cancer. Agents that induce apoptosis may inhibit tumor growth and provide therapeutic benefit. In a recent study, the authors found that certain homeopathic treatments produced anticancer effects in an animal model. In this study, the authors examined the immunomodulating and apoptotic effects of these remedies. MATERIALS AND METHODS: The authors investigated the effect of a homeopathic treatment regimen containing Conium maculatum, Sabal serrulata, Thuja occidentalis, and a MAT-LyLu Carcinosin nosode on the expression of cytokines and genes that regulate apoptosis. This was assessed in prostate cancer tissues, extracted from animals responsive to these drugs, using ribonuclease protection assay or reverse transcription polymerase chain reaction. RESULTS: There were no significant changes in mRNA levels of the apoptotic genes bax, bcl-2, bcl-x, caspase-1, caspase-2, caspase-3, Fas, FasL, or the cytokines interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-beta, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-alpha, IL-2, and interferon-gamma in prostate tumor and lung metastasis after treatment with homeopathic medicines. CONCLUSIONS: This study indicates that treatment with the highly diluted homeopathic remedies does not alter the gene expression in primary prostate tumors or in lung metastasis. The therapeutic effect of homeopathic treatments observed in the in vivo experiments cannot be explained by mechanisms based on distinct alterations in gene expression related to apoptosis or cytokines. Future research should explore subtle modulations in the expression of multiple genes in different biological pathways.

Homeopathic medicines do not alter growth and gene expression in prostate and breast cancer cells in vitro.

BACKGROUND: Homeopathy is an alternative medical system practiced in all parts of the world. Although several theories are proposed to explain the mechanisms of action, none are scientifically verified. In this study, the authors investigate the effect of selected homeopathic remedies often used to treat prostate and breast cancer. MATERIALS AND METHODS: The authors investigated the effect of the homeopathic medicines Conium maculatum, Sabal serrulata, Thuja occidentalis, Asterias, Phytolacca, and Carcinosin on prostate and breast cancer cell (DU-145, LNCaP, MAT-LyLu, MDA-MB-231) growth and on gene expression that regulates apoptosis, using MTT and multiprobe ribonuclease protection assay. RESULTS: None of the homeopathic remedies tested in different potencies produced significant inhibitory or growth-promoting activity in either prostate or breast cancer cells. Also, gene expression studies by ribonuclease protection assay produced no significant changes in mRNA levels of bax, bcl-2, bcl-x, caspase-1, caspase-2, caspase-3, Fas, or FasL after treatment with homeopathic medicines. CONCLUSIONS: The results demonstrate that the highly diluted homeopathic remedies used by homeopathic practitioners for cancer show no measurable effects on cell growth or gene expression in vitro using currently available methodologies.

Inhibition of tumor angiogenesis by Brahma Rasayana (BR).

The current therapy for prostate cancer includes radical prostatectomy, radiation therapy and hormonal ablation. Chemotherapy also provides beneficial results for some patients with advanced prostate cancer but with several harmful side effects. Hence there is a need to identify and develop alternate therapies, which can reduce the disease progression with minimal or few side effects. Earlier studies from our laboratory have shown that a Polyherbal mixture, Brahma Rasayna (BR) rich in anti-oxidant principles has a potential to be an anti-tumor agent. BR treatment of MAT-LyLu cell inoculated Copenhagen rats resulted in a decrease of palpable tumor incidence, delay in tumor occurrence and lower mean tumor volumes. Also, a significant reduction in tumor weight and lung metastasis was observed in BR treated animals in comparison to untreated controls. In the present study, we focused to examine the effect of BR on angiogenesis and regulation of molecular markers involved in angiogenesis using in-vivo and in-vitro models. BR treatment showed a significant reduction in Factor VIII expression compared to control indicating reduced angiogenesis. BR treated tumor specimens showed a decrease in the pro-angiogenic factors like VEGF, MMP-9 and MMP-2. Methanolic extract of BR was found to inhibit the proliferation, tube formation, cell migration and attachment of HUVEC on matrigel in a dose dependant manner. These findings suggest the possible mechanism(s) of action of BR in the reduction of tumor growth and metastatic spread.

Protective effect of a polyherbal preparation, Brahma rasayana against tumor growth and lung metastasis in rat prostate model system.

Rasayanas are a group of herbal formulations and are used to improve health of the body. Recent studies have demonstrated the immunostimulatory and antioxidant activities of some of these rasayanas and their usefulness in tumor regression. The objective of the study is to evaluate Brahma rasayana for the inhibition of tumor development and prevention of metastasis in vivo using Copenhagen rats and MAT-LyLu cell model system. Copenhagen rats injected with MAT-LyLu cells were treated with Brahma rasayana once daily. This treatment was followed from the second day of cell inoculation until the end of the experiment. The study comprises a comparison of survival time, body weight, tumor incidence, tumor size, tumor weight, histopathological examination of the lung metastasis and serum testosterone levels between rasayana treated and control animals. Brahma rasayana treatment resulted in a 25-37% decrease in palpable tumor incidence, a delay of 1-2 weeks in the tumor occurrence, lower mean tumor volumes, by as much as 14-35% and significant reduction in tumor weight and lung metastasis in comparison to untreated controls. The Ayurvedic poly herbal preparation, Brahma rasayana may play a beneficial role in preventing tumor incidence, tumor growth and metastatic spread. These are inexpensive preparations that have little or no adverse side effects with a potential as lead chemopreventive compounds and which might prove useful for the treatment of disorders such as human prostate cancer.

Differential regulation of cytokines and transcription factors in liver by curcumin following hemorrhage/resuscitation.

Inflammatory cytokines interleukin 1 (IL-1), IL-2, IL-6, and tumor necrosis factor-alpha (TNF-alpha) have been recognized as important mediators of pathophysiological and immunological events associated with shock. These inflammatory events after hemorrhage and resuscitation are characterized by the activation of transcription regulators such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). Curcumin, an anti-inflammatory remedy used in Indian medicine, is known to suppress NF-kappaB and AP-1 activation and also to reduce ischemia-reperfusion injuries in animal models. Therefore, the aim of this study was to determine whether administration of curcumin before hemorrhagic shock has any salutary effects on cytokines and the redox-sensitive transcription factors NF-kappaB and AP-1. mRNA levels of IL-1alpha, IL-1beta, IL-2, IL-6, IL-10, and TNF-alpha were determined by reverse transcriptase-polymerase chain reaction in rat livers collected at 2 and 24 h after hemorrhage/resuscitation. The effect of curcumin on the activation of NF-kappaB and AP-1 was determined by electrophoretic mobility shift assays. Significant increases in the levels of liver cytokines IL-1alpha, IL-1beta, IL-2, IL-6, and IL-10 were observed in the 2-h posthemorrhage/resuscitation group compared with sham animals. In contrast, oral administration of curcumin for 7 days followed by hemorrhage/resuscitation regimen resulted in significant restoration of these cytokines to depleted levels, and, in fact, IL-1beta levels were lower than sham levels. Also, the 24-h postresuscitation group showed similar patterns with some exceptions. NF-kappaB and AP-1 were differentially activated at 2 and 24 h posthemorrhage and were inhibited by curcumin pretreatment. Serum aspartate transaminase estimates indicate decreased liver injury in curcumin-pretreated hemorrhage animals. These results suggest that protection against hemorrhage/resuscitation injury by curcumin pretreatment may result from the inactivation of transcription factors involved and regulation of cytokines to beneficial levels.

Low-Dose Cadmium Exposure Reduces Human Prostate Cell Transformation in Culture and Up-Regulates Metallothionein and MT-1G mRNA.

Chronic low-level exposure to environmental toxins, including cadmium (Cd), is a growing problem in the industrialized world. One promising strategy for protection from these toxins is the use of low-dose exposure of environmental chemicals to induce cell tolerance and recovery, a phenomenon known as "protective hormesis". Hormetic [low-dose stimulatory] effects occur in a variety of systems and with a number of chemicals. Cd is a potent carcinogen in rodents and has also been linked to human lung and prostate cancers. In the present study, we have evaluated the protective effects of low and ultra-low dose, long-term Cd exposure in the normal human prostate cells, RWPE-1. Cells were exposed to low and ultra-low doses (0, 0 (S(-36)), 10(-6), 10(-7), 10(-18), 10(-21), 10(-32), or 10(-36)M) of Cd for 20 weeks followed by treatment with 10(-5)M Cd for another 8 weeks. Continuous exposure of RWPE-1 cells to 10(-5)M Cd results in malignant transformation. However, cells pretreated with low and ultra-low doses of Cd had delayed transformation compared with controls. In addition, the number of transformed cell mounds was lower in pretreated cells indicating that low and ultra-low dose exposure had protective effects against high-dose Cd induced carcinogenesis. The expression of metallothionein (MT), the primary Cd detoxification protein, was induced by low-dose exposure to Cd and maintained during the 20 weeks. In addition, MT-1G mRNA was up-regulated 2- to 3-fold by low-dose and ultralow-dose Cd exposures and may be the mechanism of protective hormesis in this model. MT-1G mRNA might also serve as a biological indicator of very low-dose environmental Cd exposure.

Curcumin differentially regulates TGF-beta1, its receptors and nitric oxide synthase during impaired wound healing.

Wound healing is a highly ordered process, requiring complex and coordinated interactions involving peptide growth factors of which transforming growth factor-beta (TGF-beta) is one of the most important. Nitric oxide is also an important factor in healing and its production is regulated by inducible nitric oxide synthase (iNOS). We have earlier shown that curcumin (diferuloylmethane), a natural product obtained from the plant Curcuma longa, enhances cutaneous wound healing in normal and diabetic rats. In this study, we have investigated the effect of curcumin treatment by topical application in dexamethasone-impaired cutaneous healing in a full thickness punch wound model in rats. We assessed healing in terms of histology, morphometry, and collagenization on the fourth and seventh days post-wounding and analyzed the regulation of TGF-beta1, its receptors type I (tIrc) and type II (tIIrc) and iNOS. Curcumin significantly accelerated healing of wounds with or without dexamethasone treatment as revealed by a reduction in the wound width and gap length compared to controls. Curcumin treatment resulted in the enhanced expression of TGF-beta1 and TGF-beta tIIrc in both normal and impaired healing wounds as revealed by immunohistochemistry. Macrophages in the wound bed showed an enhanced expression of TGF-beta1 mRNA in curcumin treated wounds as evidenced by in situ hybridization. However, enhanced expression of TGF-beta tIrc by curcumin treatment observed only in dexamethasone-impaired wounds at the 7th day post-wounding. iNOS levels were increased following curcumin treatment in unimpaired wounds, but not so in the dexamethasone-impaired wounds. The study indicates an enhancement in dexamethasone impaired wound repair by topical curcumin and its differential regulatory effect on TGF-beta1, it's receptors and iNOS in this cutaneous wound-healing model.