Targeting Brain Tumors with Nanomedicines: Overcoming Blood Brain Barrier Challenges.

BACKGROUND: This review elucidates ongoing research, which show improved delivery of anticancer drugs alone and/ or enclosed in carriers collectively called nanomedicines to cross the BBB/ BTB to kill tumor cells and impact patient survival. We highlighted various advances in understanding the mechanism of BTB function that has an impact on anticancer therapeutics delivery. We discussed latest breakthroughs in developing pharmaceutical strategies, including nanomedicines and delivering them across BTB for brain tumor management and treatment. METHODS: We performed an extensive literature search and highlighted important studies on the regulation of BTB permeability with respect to nanotech-based nanomedicines for targeted treatment of brain tumors. We have reviewed research articles that describe the development of specialized molecules and nanospheres, which carry payload of anticancer agents to brain tumor cells across the BBB/ BTB and avoid drug efflux systems. We highlighted research on the identification and development of targeted anti-cancer drug delivery to brain tumors. In addition, we discussed multimeric molecular therapeutics and nanomedicines that were encapsulated in nanospheres for treatment and monitoring of brain tumors. RESULTS: In this context, we quoted our research on large conductance calcium-activated potassium channels (BKCa) and ATP-dependent potassium channels (KATP) as portals of enhanced antineoplastic drugs delivery. We showed that several innovative drug delivery agents such as liposomes, polymeric nanoparticles, dendrimers and many such tools can be utilized to improve anticancer drugs and nanomedicines across the BTB to reach brain tumor cells. CONCLUSION: This review might interest both academic and drug company scientists involved in drug delivery to brain tumors. We further seek to present evidence that BTB modulators can be clinically developed as combination drug or/ and as stand-alone anticancer drugs. Eventually, it is expected that unrelenting effort from the scientific community in developing novel drug delivery methods should increase the survival rate of brain tumor patients, which is dismally low presently.

Targeting potassium channels for increasing delivery of imaging agents and therapeutics to brain tumors.

Every year in the US, 20,000 new primary and nearly 200,000 metastatic brain tumor cases are reported. The cerebral microvessels/capillaries that form the blood-brain barrier not only protect the brain from toxic agents in the blood but also pose a significant hindrance to the delivery of small and large therapeutic molecules. Different strategies have been employed to circumvent the physiological barrier posed by blood-brain tumor barrier (BTB). Studies in our laboratory have identified significant differences in the expression levels of certain genes and proteins between normal and brain tumor capillary endothelial cells (ECs). In this study, we validated the non-invasive and clinically relevant dynamic contrast enhancing-magnetic resonance imaging (DCE-MRI) method with invasive, clinically irrelevant but highly accurate quantitative autoradiography method using rat glioma model. We also showed that DCE-MRI metric of tissue vessel perfusion-permeability is sensitive to changes in blood vessel permeability following administration of calcium-activated potassium (BKCa) channel activator NS-1619. Our results show that human gliomas and brain tumor ECs that overexpress BKCa channels can be targeted for increased BTB permeability for MRI enhancing agents to brain tumors. We conclude that monitoring the outcome of increased MRI enhancing agents' delivery to microsatellites and leading tumor edges in glioma patients would lead to beneficial clinical outcome.

Epigenetic regulation of microRNA genes and the role of miR-34b in cell invasion and motility in human melanoma.

Invasive melanoma is the most lethal form of skin cancer. The treatment of melanoma-derived cell lines with 5-aza-2'-deoxycytidine (5-Aza-dC) markedly increases the expression of several miRNAs, suggesting that the miRNA-encoding genes might be epigenetically regulated, either directly or indirectly, by DNA methylation. We have identified a group of epigenetically regulated miRNA genes in melanoma cells, and have confirmed that the upstream CpG island sequences of several such miRNA genes are hypermethylated in cell lines derived from different stages of melanoma, but not in melanocytes and keratinocytes. We used direct DNA bisulfite and immunoprecipitated DNA (Methyl-DIP) to identify changes in CpG island methylation in distinct melanoma patient samples classified as primary in situ, regional metastatic, and distant metastatic. Two melanoma cell lines (WM1552C and A375 derived from stage 3 and stage 4 human melanoma, respectively) were engineered to ectopically express one of the epigenetically modified miRNA: miR-34b. Expression of miR-34b reduced cell invasion and motility rates of both WM1552C and A375, suggesting that the enhanced cell invasiveness and motility observed in metastatic melanoma cells may be related to their reduced expression of miR-34b. Total RNA isolated from control or miR-34b-expressing WM1552C cells was subjected to deep sequencing to identify gene networks around miR-34b. We identified network modules that are potentially regulated by miR-34b, and which suggest a mechanism for the role of miR-34b in regulating normal cell motility and cytokinesis.

The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.

The identification of cancer-associated long noncoding RNAs (lncRNAs) and the investigation of their molecular and biological functions are important to understand the molecular biology of cancer and its progression. Although the functions of lncRNAs and the mechanisms regulating their expression are largely unknown, recent studies are beginning to unravel their importance in human health and disease. Here, we report that a number of lncRNAs are differentially expressed in melanoma cell lines in comparison to melanocytes and keratinocyte controls. One of these lncRNAs, SPRY4-IT1 (GenBank accession ID AK024556), is derived from an intron of the SPRY4 gene and is predicted to contain several long hairpins in its secondary structure. RNA-FISH analysis showed that SPRY4-IT1 is predominantly localized in the cytoplasm of melanoma cells, and SPRY4-IT1 RNAi knockdown results in defects in cell growth, differentiation, and higher rates of apoptosis in melanoma cell lines. Differential expression of both SPRY4 and SPRY4-IT1 was also detected in vivo, in 30 distinct patient samples, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic melanoma. The elevated expression of SPRY4-IT1 in melanoma cells compared to melanocytes, its accumulation in cell cytoplasm, and effects on cell dynamics, including increased rate of wound closure on SPRY4-IT1 overexpression, suggest that the higher expression of SPRY4-IT1 may have an important role in the molecular etiology of human melanoma.

7, 8-diacetoxy-4-methylcoumarin induced cell death in human tumor cells is influenced by calreticulin.

Calreticulin (CRT), an endoplasmic reticulum resident protein demonstrates transacetylase activity in presence of 7, 8 diacetoxy-4-methyl coumarin (DAMC) in vitro. To investigate the possible role of CRT and DAMC mediated protein acetylation in cells, we investigated the effects of DAMC in tumor cells with different levels of CRT. DAMC was more toxic (clonogenicity, metabolic viability and proliferation) to human glioma cells (BMG-1) expressing low endogenous CRT level as compared to head and neck carcinoma cells (KB) with a high CRT level. The cytotoxicity was accompanied by loss of mitochondrial membrane potential in both the cells, which correlated with corresponding changes in the levels of pro-apoptotic (Bax) and anti-apoptotic (NFkB) regulators. Manipulation of CRT protein level in KB cells by application of small RNA interference enhanced the sensitivity by four folds while over expression of CRT in BMG-1 cells reduced their sensitivity to DAMC by ~20% strongly suggesting the influence of CRT on DAMC induced cytotoxicity. The partial rescue of CROE cells from DAMC induced toxicity was accompanied by changes in NFkB levels and over all protein acetylation status, besides increase in the NADPH-cytochrome c reductase activity related to its well known antioxidant property. Since CRT is over-expressed in cancer cells, which are generally resistant to radio- and chemotherapy; targeting CRT transacetylase system, may be an attractive approach for increasing the efficacy of anticancer therapies.

The regulation of miRNA-211 expression and its role in melanoma cell invasiveness.

The immediate molecular mechanisms behind invasive melanoma are poorly understood. Recent studies implicate microRNAs (miRNAs) as important agents in melanoma and other cancers. To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs. Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic. The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211. In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1. Mutating the miR-211 binding site seed sequences at the KCNMA1 3'-UTR abolished target cleavage. KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels. Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines. An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis. The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression. MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211's negative post transcriptional effects on the KCNMA1 transcript. Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well as the dependence of this miRNA's expression on MITF activity, establishes miR-211 as an important regulatory agent in human melanoma.

Short-term exposure of multicellular tumor spheroids of a human glioma cell line to the glycolytic inhibitor 2-deoxy-D-glucose is more toxic than continuous exposure.

The glycolytic inhibitor 2-deoxy-D-glucose (2-DG) has been used as a therapeutic agent and as an adjuvant in cancer therapy with either weekly fractions of the treatment or daily administration. While the weekly fraction has often been found to be nontoxic and effective, other treatment regimes are tolerated to a relatively lesser extent. It was therefore, considered worthwhile to investigate the efficacy of short- and long-term exposure of tumor cells to 2-DG under the controlled conditions. Seven-day-old MTS were exposed to 2-DG (5 mM, equimolar to glucose concentration in media) for different time intervals (30 min to 24 h) trypsinized and plated for clonogenicity. Alternatively, spheroids were grown either continuously in the presence of 2-DG or were treated with 2-DG for 2 h (short-term exposure) and grown in 2-DG-free media for 21 days and assessed for spheroid growth, cell viability, apoptosis, cytogenetic damage, mitochondrial status, and oxidative stress. Exposure of spheroids to 2-DG for 2-4 h induced 30% cell death (SF 0.70) while, a 24-h exposure resulted in only a marginal decrease in clonogenicity (SF 0.95). Furthermore, the spheroids disintegrated completely by 28 days in the case of 2-h exposure to 2-DG, while spheroids grown continuously in the presence of 2-DG repopulated. The cytotoxicity following short-term exposure of MTS to 2-DG was primarily due to the induction of apoptosis revealed by morphological features as well as flow cytometric analysis of the DNA content. Interestingly however, cytogenetic damage (micronuclei induction) was observed in spheroids that were continuously exposed to 2-DG. Short-term exposure to 2-DG resulted in a significant increase in ROS levels and a reduction in the levels of unoxidized cardiolipin as measured by NAO suggesting the involvement of mitochondria leakiness leading to oxidative stress which, could be responsible for apoptotic cell death observed under these conditions. However, continuous exposure to 2-DG resulted in a moderate level of oxidative stress leading to the genomic instability. Preliminary studies also show that spheroids exposed continuously to 2-DG result in the development of resistance to certain chemotherapeutic drugs which could be correlated with elevated levels of mdr1. The present results suggest that a persistent down-regulation of glycolysis (as seen here with continuous exposure to 2-DG) could activate prosurvival responses besides inducing moderate levels of oxidative stress resulting in the development of resistance against therapeutic agents.

Modulation of KCa channels increases anticancer drug delivery to brain tumors and prolongs survival in xenograft model.

Most anticancer drugs fail to impact patient survival since they fail to cross the blood-brain tumor barrier (BTB) at therapeutic levels. For example, Temozolomide (TMZ) exhibits some antitumor activity against brain tumors, so does Trastuzumab (Herceptin, Her-2 inhibitor), which might be effective against Her2 neu overexpressing gliomas. Nevertheless, intact BTB and active efflux system may prevent their entry to brain tumors. Previously we have shown that potassium channel agonists increased carboplatin and Her-2 neu antibody delivery in animal glioma models. Here, we studied whether potassium channel agonist increase TMZ and Herceptin delivery across the BTB to elicit antitumor activity and increase survival in nude mice with human glial tumor. The K(Ca) channel activity and expression was also evaluated in human glioma tissues. We administered NS-1619, calcium-dependent potassium (K(Ca)) channel agonist, with [(14)C]-TMZ, and quantified TMZ delivery. The results clearly demonstrate that when given systemically both TMZ and Herceptin do not cross the BTB in significant amounts, however, NS-1619 co-infusion with [(14)C]-TMZ and Herceptin resulted in enhanced drug delivery to brain-tumor cells. The combination treatment of TMZ and Herceptin also showed improved antitumor effect which was more prominent than that of either treatment alone in increasing the survival in mice with brain tumor, when co-infused with K(Ca) channel agonists. In conclusion, K(Ca) channel agonists may benefit brain tumor patients by increasing anti-neoplastic agent's delivery to brain tumors. A clinical outcome of this research is the discovery of a novel drug delivery system that circumvents the BBB/BTB to benefit brain tumor patients.

Role of KCNMA1 gene in breast cancer invasion and metastasis to brain.

BACKGROUND: The prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, large-conductance calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene that encodes for the pore-forming alpha-subunit of BKCa channels in breast cancer metastasis and invasion. METHODS: We performed Global exon array to study the expression of KCNMA1 in metastatic breast cancer to brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of BKCa channel protein in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether BKCa channel inhibition attenuates breast tumor invasion and metastasis using KCNMA1 knockdown with siRNA and biochemical inhibition with Iberiotoxin (IBTX). RESULTS: The Global exon array and RT-PCR showed higher KCNMA1 expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased BKCa channel activity, leading to greater invasiveness and transendothelial migration, both of which could be attenuated by blocking KCNMA1. CONCLUSION: Determining the relative abundance of BKCa channel expression in breast cancer metastatic to brain and the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy.

Endogenous and induced oxidative stress in multi-cellular tumour spheroids: implications for improving tumour therapy.

The endogenous oxidative stress in tumours is determined by the status of mitochondrial, metabolic, oxygen (hypoxia) and inherent enzymatic as well as non-enzymatic antioxidant defense systems, which influence tumour growth and respond to anticancer therapeutics. Induced oxidative stress is one of the important determinants of the outcome of treatment with certain chemotherapeutic drugs and ionizing radiation. The mild to moderate levels of reactive oxygen species (ROS) have often been found to trigger prosurvival responses, thereby contributing to the resistance against therapy. The higher levels of ROS stimulate multiple death pathways viz. typical and atypical apoptosis, necrosis etc, thereby enhancing the therapeutic efficiency. Therefore, approaches employing therapeutic agents that generate ROS efficiently in the tumour cells and enhance the antioxidant defense system in the normal cells could significantly enhance the therapeutic gain. Multi-cellular tumour spheroids (MCTS) offer an excellent in vitro system that mimics endogenous oxidative stress often observed in tumours, arising due to a number of factors (gradients of oxygen and nutrients, altered intercellular interaction and tumour necrosis factor), besides antioxidant defense systems similar to tumours in vivo. More importantly, MCTS resemble tumours in vivo with reference to the induced oxidative stress related responses, particularly following combinations of certain chemotherapeutic drugs and metabolic inhibitors and differs significantly from the responses in monolayer cultures. Therefore, MCTS appear to be excellent in vitro models, ideally suited for developing novel therapies that are based on the generation of oxidative stress in tumours. The present review provides a modest account on the utility of MCTS in understanding the role of oxidative stress in treatment-induced responses of tumours for designing therapies and therapeutics.

Stress response of a p53 homologue in the radioresistant Sf9 insect cells.

PURPOSE: To investigate homology and stress response of p53 (a 53 kDa tumor suppressor protein) orthologue in Sf9 Lepidopteran insect cell line that exhibits very high radioresistance. MATERIALS AND METHODS: Western immunoblotting, immunoprecipitation, degenerate RT-PCR (reverse transcription-polymerase chain reaction), electrophoretic gel mobility shift assay, flow cytometry and immuno-fluorescence microscopy were used for characterizing structural and functional features of Sfp53 (Spodoptera frugiperda p53) in gamma-irradiated or etoposide-treated Sf9 insect and BMG-1 (brain malignant glioma) human cells. Cells were pre-treated with caffeine for inhibiting ATM/ATR (ataxia-telangiectasia mutated protein/ATM and Rad-3-related protein) activation, wherever required. RESULTS: A 47-49 kDa protein band was observed with antibodies against three different epitopes, demonstrating conservation of respective domains in Sfp53. Immunoprecipitation also yielded similar-sized protein. Degenerate RT-PCR resulted in product of same size in both cell lines. Similar gel mobility shift of p53-binding oligonucleotide with BMG-1 and Sf9 cell lysates indicated analogous transcriptional activity of Sfp53. Constitutive Sfp53 level was higher than hp53 (human p53) and showed primarily cytoplasmic localization. Radiation-induced accumulation was considerably less in Sf9 even as an analogous ATM/ATR-dependent nuclear translocation was observed following gamma-irradiation and etoposide. CONCLUSIONS: A smaller-sized Sfp53 orthologue shows highly conserved native structure with DNA-binding, N-terminus and C-terminus domains, and has analogous p53 transcriptional activity. While its nuclear translocation and ATM/ATR dependence were similar to hp53, the cytoplasmic localization and subdued accumulation following gamma-irradiation indicate functional differences from human cells.

Activation of KATP channels increases anticancer drug delivery to brain tumors and survival.

Several anticancer drugs are ineffective against brain tumor and do not impact patient survival because they fail to cross the blood-brain tumor barrier (BTB) effective levels. One such agent temozolomide is commonly used in brain tumor patients, which works better when combined with radiation or other anticancer agents. Likewise, trastuzumab (Herceptin, Her-2 inhibitor), which might be effective against Her2/neu over expressing gliomas may work well when combined with temozolomide. Nonetheless, both drugs do not cross the BTB to significantly impact patient survival. Beforehand we showed that potassium channel agonists when intracarotidly administered increased carboplatin and Her-2 antibody delivery in animal glioma models by triggering formation of brain vascular endothelial transcytotic vesicles. In this study, we investigated whether, intravenously administered, ATP-sensitive potassium channel (K(ATP)) activator (minoxidil sulfate; MS) increases temozolomide and Herceptin delivery to brain tumors to induce anti-tumor activity and increase survival in nude mice with Glioblastoma multiforme (GBM) cells. The results clearly demonstrate that when given intravenously temozolomide crosses BTB at a relatively low amount while Herceptin failed to cross the BTB. However, MS co-infusion with [(14)C]-temozolomide or fluorescently labeled-Herceptin resulted in improved and selective drug delivery to brain tumor. We also showed that combination treatment with temozolomide and Herceptin has enhanced anti-tumor effect which was more prominent than that of either treatment alone in increasing the survival in mice with GBM when co-infused with MS. Therefore, brain tumor patients may be benefited when anti-neoplastic agent delivery is increased selectively to the brain tumors using KATP channel agonists.

Differential mechanisms of radiosensitization by 2-deoxy-D-glucose in the monolayers and multicellular spheroids of a human glioma cell line.

In vitro studies using monolayer cultures of human tumor cell lines have shown that 2-DG selectively inhibits energy-dependent DNA repair and cellular recovery processes in cancer cells. However, monolayer cultures differ greatly from the complex environmental conditions generated in solid tumors that develop inhomogeneous hypoxic and necrotic regions. In contrast, multicellular spheroids mimic heterogeneous cellular behavior and the consequent functional characteristics of in vivo solid tumors, and serve as important in vitro model to investigate tumor biology and responses to potential therapeutic agents. The present study compares the radiomodification by 2-DG in monolayer cultures and spheroids of a human glioma cell line (BMG-1) to gain insight into the effects in solid tumors. In spheroids, the glucose consumption (2.1 p mole/cell/h) and lactate production (3.67 p mole/cell/h) was nearly 2-3 fold higher than in monolayer cells (0.83 and 1.43 p mole/cell/h respectively). Presence of 2-DG (5 mM) for 2-4 h inhibited the glucose usage and lactate production by 70% in spheroids, while a 35% reduction was observed in monolayer cells. Under these conditions, 2-DG drastically enhanced the radiation-induced cell death of spheroids (by 2-3 folds); while a 40% increase was observed in monolayer cells. Radiosensitization by 2-DG in monolayer cells was primarily due to an increase in mitotic death (23%) linked to cytogenetic damage (micronuclei), whereas a profound induction of apoptosis (40%) accounted for the sensitization in spheroids. Although the Bcl-2 and Bax levels were significantly higher in spheroids, Bcl-2/Bax ratio was similar in monolayers and spheroids. Comet assay revealed a late onset of DNA breaks in the presence of 2- DG following irradiation only in spheroids, which corroborated well with the late onset of oxidative stress. 2-DG did not induce a significant cell cycle delay in monolayers, while a transient G(2) delay was apparent in spheroids.

Establishment and characterization of multicellular spheroids from a human glioma cell line; Implications for tumor therapy.

BACKGROUND: Multicellular spheroids, an appropriate in vitro system for simulating 3-D tumor micro-milieu can be used for evaluating and predicting tumor response to therapeutic agents including metabolic inhibitors. However, detailed understanding of the nature, distribution and sensitivity/responses of cellular sub-populations to potential therapeutic agents/strategies is required for using this unique model with optimal precision. Spheroid characteristics may also vary considerably with the origin and type of cell line used, and thorough characterization of viable and dissociated glioma cell spheroids is not yet completely known. In order to evaluate in vivo responses of gliomas to various therapeutic strategies, especially the metabolic inhibitors capable of penetrating the blood brain barrier, we have characterized continuously growing spheroids of a human glioma cell line (BMG-1) with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress and radiation response using microscopy, flow cytometry and enzymatic assays. Spheroids were fed daily with fresh medium in order to maintain nutrient supply to outer cellular layers while hypoxia/necrosis developed in the innermost cells of enlarging spheroids. RESULTS: Volume of spheroids, fed daily with fresh medium, increased exponentially during 7-28 days of growth through three population doublings. Proportion of G1-phase cells was higher (approximately 60%) than exponentially growing monolayer cells (approximately 48%). A significant fraction of S-phase cells turned metabolically inactive (disengaged in DNA synthesis) with increasing age of the spheroids, unlike in quiescent monolayer cultures, where the fraction of S-phase cells was less than 5%. With increasing spheroid size, increasing sub-populations of cells became non-viable and entered apoptosis or necrosis revealed by Annexin-V-FITC/PI staining. PI positive (necrotic) cells were not confined to the centre of the spheroid, but distributed at certain discrete foci. Average glucose consumption and lactate production were 2-3 folds higher in viable spheroid cells compared to monolayer cells, implying a compensatory increase in glycolysis possibly due to hypoxic environment. HIF-1alpha was expressed only in spheroids and increased in an age-dependent manner, whereas c-Myc (known to induce apoptosis in glucose-deprived cells) levels were three times higher than monolayer cells. Mitochondrial mass and activity decreased significantly during first 14 days of growth but increased with age, and were not associated with increase in ROS levels. Bcl-2 and Bax levels were higher (approximately 2 folds) than monolayers, while the ratio (Bcl/Bax) remained unaltered. Radiation-induced oxidative stress was considerably less in spheroids as compared to monolayers, and corresponded well with increase in radioresistance demonstrated by the clonogenic assay, similar to hypoxia induced radioresistance observed in tumors. CONCLUSION: Development of S-negative cells and reduced endogenous and radiation-induced ROS coupled with higher levels of anti (Bcl2) as well as pro (Bax) apoptotic regulators observed in spheroids suggest the intricate/complex nature of endogenous as well as induced stress resistance that could exist in tumors, which contribute to the treatment resistance.

Multicellular spheroids as an in vitro model in experimental oncology: applications in translational medicine.

Multicellular tumour spheroids (MCTS), the best-described three-dimensional tumour model, offers an excellent in vitro screening system, which to a great extent mimics the microenvironment prevailing in the tumour tissue, supporting studies on such tumour specific processes such as angiogenesis, invasion and metastasis and the assessment of responses to various therapies as well as their underlying mechanisms. Developed as an alternative in vitro model to monolayer cultures, nearly 30 years ago, their role as part of high-throughput cell-based assay system in drug discovery is gaining considerable importance. The distinct possibility of establishing MCTS from primary tumour cells and co-culturing with different normal cells, namely stromal fibroblasts, endothelial cells and cells of hemopoietic as well as immune system lineage, enhance the value of MCTS in cancer research and drug development. Here, the authors present a brief overview on the present status on the application of multicellular spheroids in experimental oncology, particularly from the view point of cancer therapy and drug development.

Heterogeneity in the radiosensitizing effects of the DNA ligand hoechst-33342 in human tumor cell lines.

The AT specific minor grove DNA binding ligands bisbenzimidazole derivatives like hoechst-33342 and hoechst-33258 which scavenge free radicals and stabilize macromolecular structure have been shown to afford radioprotection by reducing the induction of DNA damage. However, their ability to inhibit topoisomerases I & II, which play important roles in damage response pathways including DNA repair can enhance radiation damage under certain conditions. Since pool sizes of the topoisomerases differ not only between normal and tumor cells, but also among different tumors, it is anticipated that radiosensitization by hoechst-33342 can vary among tumors. The present studies were, therefore, undertaken to verify this proposition in human glioma (BMG-1 &U-87) and squamous carcinoma (4197 &4451) cell lines which differ in their biological behavior (ploidy, p53, cyclins, bcl, bax etc). Isotoxic concentrations of hoechst-33342 (IC50 i.e producing 50% cell kill) administered immediately following irradiation resulted in the radiosensitization of all cell lines, with a 4&7 fold increase in the cell death (loss of clonogenic cell survival) in U-87&BMG-1 and a 3 fold increase in 4197 &4451 cells. Growth inhibition and increase in cytogenetic damage (micronuclei formation) as well as delayed apoptosis observed under these conditions corroborated well with the enhanced cell death. The ligand induced a significant cell cycle delay, particularly in the late S and G2 phases of BMG-1, U-87 and 4197 cells, while no significant changes could be observed in 4451 cells. Higher endogenous levels of cyclin B1 found in both the glioma cell lines, was enhanced further by the ligand as compared to the squamous carcinoma cells. These results clearly demonstrate that the radiosensitizing effects of the ligand are indeed heterogeneous among different human tumor cell lines. The radiaosensitization is p53 independent and accompanied by enhanced mitotic death (linked to cytogenetic damage) as well as induction of cyclin B1 mediated apoptosis.

Inhibitors of topoisomerases as anticancer drugs: problems and prospects.

DNA topoisomerases, which solve topological problems associated with various DNA transactions, are the targets of many therapeutic agents. Various topoisomerase inhibitors especially, topo-poisons, camptothecin (topo-I) and etoposide (topo-II) are some of the drugs that are used in the current treatment protocols, particularly for the treatment of leukemia (AML, ALL etc). However, tumor resistance, normal and non-specific tissue cytotoxicity are the limitations for successful development of these drugs as one of the primary therapeutic agents for the treatment of tumors in vitro. This brief review presents the current understanding about cytotoxicity development and outlines various approaches to overcome the limitations for enhancing the efficacy of topo-poison based anticancer drugs.

2-deoxy-D-glucose enhances the cytotoxicity of topoisomerase inhibitors in human tumor cell lines.

Resistance of tumors due to restricted drug accumulation and reversal of DNA lesions in tumor cells as well as normal tissue toxicity limit the efficacy of topoisomerase inhibition based anticancer drugs. It has been proposed that selective inhibition of energy dependent repair processes and enhanced retention of drug in cancer cells can significantly improve the therapeutic efficacy. The purpose of these studies was to verify this proposition by investigating the effects of 2-deoxy-D-glucose (2-DG) an inhibitor of the glycolytic ATP production on the cytotoxicity of certain topoisomerase inhibitors in human tumor cell lines. Human glioma (BMG-1 and U-87) and squamous carcinoma (4197 and 4451) cell lines were investigated with topo-poisons like etoposide (topo II inhibitor), camptothecin (topo I inhibitor) and hoechst-33342 (topo I and II inhibitor). DNA damage induction (halo assay), cell survival (macro colony assay), cytogenetic damage (micronuclei) and apoptosis (morphological analysis) were investigated. Presence of 2-DG for 2 h following exposure to the topoisomerase inhibitors enhanced the cell death (macro colony assay) in a concentration dependent manner and a 2-3-fold increase was observed at 5 mM (equimolar with glucose). Halo assay revealed that 2-DG inhibited the reversal of cleavable complex leading to the accumulation of DNA strand breaks. Under these conditions 2-DG enhanced the drug-induced micronuclei formation by nearly 2 folds with etoposide and hoechst-33342 and a 4-fold increase in delayed apoptosis was observed in case of etoposide. These results clearly demonstrate that presence of 2-DG for a few hours following exposure to topo-inhibitors enhances the cytotoxicity, primarily by increasing the cytogenetic damage.

Influence of phenyl ring disubstitution on bisbenzimidazole and terbenzimidazole cytotoxicity: synthesis and biological evaluation as radioprotectors.

DNA minor groove binders, Hoechst 33258 and Hoechst 33342, have been reported to protect against radiation-induced DNA-strand breakage, but their mutagenicity and cytotoxicity limit their use as protectors of normal tissue during radiotherapy and as biological radioprotectors during accidental radiation exposure. On the basis of these observations, two new nontoxic disubstituted benzimidazoles were synthesized, one having two methoxy groups (5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl]benzimidazole, 5) and another having a methoxy and a hydroxyl group (5-(4-methylpiperazin-1-yl)-2-[2'[2''-(4-hydroxy-3-methoxyphenyl)-5' '-benzimidazolyl]-5'-benzimidazolyl]benzimidazole, 6) ortho to each other on the phenyl ring. The radiomodifying effects of these nontoxic ligands were investigated with a human glioma cell line exposed to low linear energy transfer radiation by determining cell survival and cell proliferation compared with effects of the parent compound, Hoechst 33342. Cytotoxicity assayed by analyzing clonogenicity, cell growth, and metabolic viability showed that both 5 and 6 were nontoxic at 100 microM after 72 h of exposure, whereas Hoechst 33342 resulted in lysis of 77% of these cells in 24 h. Macrocolony assay (clonogenicity) showed that 73%, 92%, and 10% of the cells survived when treated with 100 microM 5, 6, and Hoechst 33342, respectively. Both 5 and 6 did not affect the growth of BMG-1 cells. At 10 microM, 5 and 6 showed 82% and 37% protection against radiation-induced cell death (macrocolony assay) while 100% protection was observed against growth inhibition. Disubstitution of the phenyl ring has not only reduced cytotoxicity but also enhanced DNA-ligand stability, conferring high degree of radioprotection.

Low-dose radiation hypersensitivity in human tumor cell lines: effects of cell-cell contact and nutritional deprivation.

The hyper-radiosensitivity at low doses recently observed in vitro in a number of cell lines is thought to have important implications for improving tumor radiotherapy. However, cell-cell contact and the cellular environment influence cellular radiosensitivity at higher doses, and they may alter hyper-radiosensitivity in vivo. To confirm this supposition, we investigated the effects of cell density, multiplicity and nutritional deprivation on low-dose hypersensitivity in vitro. Cell survival in the low-dose range (3 cGy to 2 Gy) was studied in cells of two human glioma (BMG-1 and U-87) and two human oral squamous carcinoma (PECA-4451 and PECA-4197) lines using a conventional macrocolony assay. The effects of cell density, multiplicity and nutritional deprivation on hyper-radiosensitivity/induced radioresistance were studied in cells of the BMG-1 cell line, which showed prominent hypersensitivity and induced radioresistance. The induction of growth inhibition, cell cycle delay, micronuclei and apoptosis was also studied at the hyper-radiosensitivity-inducing low doses. Hyper-radiosensitivity/induced radioresistance was evident in the cells of all four cell lines to varying extents, with maximum sensitivity at 10-30 cGy, followed by an increase in survival up to 50 cGy-1 Gy. Both the glioma cell lines had more prominent hyper-radiosensitivity than the two squamous carcinoma cell lines. Low doses inducing maximum hyper-radiosensitivity did not cause significant growth inhibition, micronucleation or apoptosis in BMG-1 cells, but a transient G(1)/S-phase block was evident. Irradiating and incubating BMG-1 cells at high density for 0 or 4 h before plating, as well as irradiating cells as microcolonies, reduced hyper-radiosensitivity significantly, indicating the role of cell-cell contact-mediated processes. Liquid holding of BMG-1 cells in HBSS + 1% serum during and after irradiation for 4 h significantly reduced hyper-radiosensitivity, suggesting that hyper-radiosensitivity may be due partly to active damage fixation processes at low doses. Therefore, our findings suggest that the damage-induced signaling mechanisms influenced by (or mediated through) cell-cell contact or the cellular environment, as well as the lesion fixation processes, play an important role in hyper-radiosensitivity. Further studies are required to determine the exact nature of the damage that triggers these responses as well as for evaluating the potential of low-dose therapy.