Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway.

BACKGROUND: Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells. RESULTS: The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage. CONCLUSION: The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro.

BACKGROUND: Riboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo. RESULTS: Results indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway. CONCLUSION: Thus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.

Importance of serum source for the in vitro replicative senescence of human bone marrow derived mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) may be used for therapeutic applications. Culture conditions such as the serum source may impact on cell quality and the onset of replicative senescence. We have examined the effect of culturing hMSCs in autologous serum (AS) versus fetal bovine serum (FBS) on factors involved in in vitro replicative senescence. hMSCs from four donors were cultured in 10% FBS or 10% AS until they reached senescence. Cells were harvested at early passage and near senescence to study factors known to be involved in cellular senescence. The number of population doublings till senescence was similar for cells cultured in FBS, but varied greatly for hMSCs cultured in AS. FBS cells accumulated in S phase of cell cycle. This could not be explained by increased expression of cell cycle inhibitor proteins. Heat shock proteins were upregulated in AS compared to FBS cells. Reactive oxygen species and nitric oxide were upregulated in senescent FBS cells. Telomeres were shorter in senescent cells, more significantly in FBS cells. The source of serum was a determinant for the time till senescence in cultured hMSC. Serum source affected aspects of cell cycle regulation and the levels of heat shock proteins. Several mechanisms are likely to be responsible for replicative senescence in hMSC. Insight into the molecular details of how serum factors impacts on these mechanisms is important for the safe use of hMSCs in clinical applications.

Modulation of DNA glycosylase activities in mesenchymal stem cells.

Adipose-tissue derived mesenchymal stem cells (AT-MSCs) are a promising tool for use in cell-based therapies. However, in vitro expansion is required to obtain clinically relevant cell numbers, and this might increase the chance of genomic instability. DNA repair is crucial for maintaining DNA integrity. Here we have compared the initial step of base excision repair in uncultured and cultured AT-MSCs by analysis of base removal activities and expression levels of relevant DNA glycosylases. Uracil, 5-hydroxyuracil and ethenoadenine removal activities were upregulated in cultured cells compared to uncultured cells. In contrast, both the 8-oxo-7,8-dihydroguanine (8-oxoG) removal activity and the concentration of 8-oxoG bases in the DNA were reduced in the cultured cells. Gene expression analysis showed no substantial changes in mRNA expression. The glycosylase activities remained stable through at least 12 passages, suggesting that DNA repair is proficient through the period required for in vitro expansion of AT-MSCs to clinically relevant numbers.

Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum.

Culture of mesenchymal stem cells (MSCs) under conditions promoting proliferation and differentiation, while supporting genomic and epigenetic stability, is essential for therapeutic use. We report here the extent of genome-wide DNA gains and losses and of DNA methylation instability on 170 cancer-related promoters in bone marrow (BM) MSCs during culture to late passage in medium containing fetal bovine serum (FBS) or autologous serum (AS). Comparative genomic hybridization indicates that expansion of BMMSCs elicits primarily telomeric deletions in a subpopulation of cells, the extent of which varies between donors. However, late passage cultures in AS consistently display normal DNA copy numbers. Combined bisulfite restriction analysis and bisulfite sequencing show that although DNA methylation states are overall stable in culture, AS exhibits stronger propensity than FBS to maintain unmethylated states. Comparison of DNA methylation in BMMSCs with freshly isolated and cultured adipose stem cells (ASCs) also reveals that most genes unmethylated in both BMMSCs and ASCs in early passage are also unmethylated in uncultured ASCs. We conclude that (i) BMMSCs expanded in AS or FBS may display localized genetic alterations, (ii) AS tends to generate more consistent genomic backgrounds and DNA methylation patterns, and (iii) the unmethylated state of uncultured MSCs is more likely to be maintained in culture than the methylated state.

Characterization of BMP signaling dependent osteogenesis using a BMP depletable avianized bone marrow stromal cell line (TVA-BMSC).

Adipogenesis, chondrogenesis and osteogenesis are BMP signaling dependent differentiation processes. However, the molecular networks operating downstream of BMP signaling to bring about these distinct fates are yet to be fully elucidated. We have developed a novel Bone Marrow Stromal Cell (BMSC) derived mouse cell line as a powerful in vitro platform to conduct such experiments. This cell line is a derivative of BMSCs isolated from a tamoxifen inducible Bmp2 and Bmp4 double conditional knock-out mouse strain. These BMSCs are immortalized and stably transfected with avian retroviral receptor TVA (TVA-BMSCs), enabling an easy method for stable transduction of multiple genes in these cells. In TVA-BMSCs multiple components of BMP signaling pathway can be manipulated simultaneously. Using this cell line we have demonstrated that for osteogenesis, BMP signaling is required only for the first three days. We have further demonstrated that Klf10, an osteogenic transcription factor which is transcribed in developing bones in a BMP signaling dependent manner, can largely compensate for the loss of BMP signaling during osteogenesis of BMSCs. TVA-BMSCs can undergo chondrogenesis and adipogenesis, and hence may be used for dissection of the molecular networks downstream of BMP signaling in these differentiation processes as well.

Layered double hydroxides as effective carrier for anticancer drugs and tailoring of release rate through interlayer anions.

Hydrophobic anticancer drug, raloxifene hydrochloride (RH) is intercalated into a series of magnesium aluminum layered double hydroxides (LDHs) with various charge density anions through ion exchange technique for controlled drug delivery. The particle nature of the LDH in presence of drug is determined through electron microscopy and surface morphology. The release of drug from the RH intercalated LDHs was made very fast or sustained by altering the exchangeable anions followed by the modified Freundlich and parabolic diffusion models. The drug release rate is explained from the interactions between the drug and LDHs along with order-disorder structure of drug intercalated LDHs. Nitrate bound LDH exhibits greater interaction with drug and sustained drug delivery against the loosely interacted phosphate bound LDH-drug, which shows fast release. Cell viability through MTT assay suggests drug intercalated LDHs as better drug delivery vehicle for cancer cell line against poor bioavailability of the pure drug. In vivo study with mice indicates the differential tumor healing which becomes fast for greater drug release system but the body weight index clearly hints at damaged organ in the case of fast release system. Histopathological experiment confirms the damaged liver of the mice treated either with pure drug or phosphate bound LDH-drug, fast release system, vis-à-vis normal liver cell morphology for sluggish drug release system with steady healing rate of tumor. These observations clearly demonstrate that nitrate bound LDH nanoparticle is a potential drug delivery vehicle for anticancer drugs without any side effect.

Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-gingerol in cervical cancer cells.

Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer.

(6)-Gingerolinduced myeloid leukemia cell death is initiated by reactive oxygen species and activation of miR-27b expression.

The natural polyphenolic alkanone (6)-gingerol (6G) has established anti-inflammatory and antitumoral properties. However, its precise mechanism of action in myeloid leukemia cells is unclear. In this study, we investigated the effects of 6G on myeloid leukemia cells in vitro and in vivo. The results of this study showed that 6G inhibited proliferation of myeloid leukemia cell lines and primary myeloid leukemia cells while sparing the normal peripheral blood mononuclear cells, in a concentration- and time-dependent manner. Mechanistic studies using U937 and K562 cell lines revealed that 6G treatment induced reactive oxygen species (ROS) generation by inhibiting mitochondrial respiratory complex I (MRC I), which in turn increased the expression of the oxidative stress response-associated microRNA miR-27b and DNA damage. Elevated miR-27b expression inhibited PPARγ, with subsequent inhibition of the inflammatory cytokine gene expression associated with the oncogenic NF-κB pathway, whereas the increased DNA damage led to G2/M cell cycle arrest. The 6G induced effects were abolished in the presence of anti-miR-27b or the ROS scavenger N-acetylcysteine. In addition, the results of the in vivo xenograft experiments in mice indicated that 6G treatment inhibited tumor cell proliferation and induced apoptosis, in agreement with the in vitro studies. Our data provide new evidence that 6G-induced myeloid leukemia cell death is initiated by reactive oxygen species and mediated through an increase in miR-27b expression and DNA damage. The dual induction of increased miR-27b expression and DNA damage-associated cell cycle arrest by 6G may have implications for myeloid leukemia treatment.

Phenotype and gene expression of human mesenchymal stem cells in alginate scaffolds.

Human mesenchymal stem cells (MSC) are popular candidates for tissue engineering. MSC are defined by their properties in two-dimensional (2D) culture systems. Cells in 2D are known to differ from their in vivo counterparts in cell shape, proliferation, and gene expression. Little is so far known about the phenotype and gene expression of cells in three-dimensional (3D) culture systems. To begin to unravel the impact of 3D versus 2D culture conditions on MSC, we have established MSC from adipose tissue and bone marrow in 3D cultures in alginate beads covalently modified with the tripeptide arginine-glycine-aspartic acid (RGD), the integrin-binding motif found in several molecules within the extracellular matrix. The MSC changed from their fibroblastoid shape (2D) to a small, compact shape when embedded in RGD alginate (3D). High viability was maintained throughout the experiment. The MSC retained expression of integrins known to bind RGD, and practically ceased to proliferate. Microarray analysis revealed that the gene expression in cells in RGD alginate was different both from the cells cultured in 2D and from prospectively isolated, uncultured MSC, but more similar to 2D cells. As alginate may be entirely dissolved, leaving the cells as single cell suspensions for various analyses, this represents a useful model for the study of cells in 3D cultures.