Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Lung carcinoma recognition by blood dielectric spectroscopy.

Lung carcinoma has become one of the malignancies whose morbidity and mortality are growing significantly. Blood is a functional body fluid, it delivers oxygen and nutrients to the other parts of the body, and it is a pathway of tumor metastasis. This paper studies the possibility of diagnosing lung carcinoma by blood dielectric spectroscopy. Comparison experiments were carried out to trace the dielectric spectroscopy of blood with and without lung carcinoma changing at different tumor stages from 100 Hz to 100 MHz. The research results show that the discrepancy of complex permittivity between rat blood with and without lung carcinoma becomes significant with tumor growing, which is worthy for clinical diagnosis.

Hematopoietic stem and progenitor cell migration after hypofractionated radiation therapy in a murine model.

PURPOSE: To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model. METHODS AND MATERIALS: Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients. RESULTS: Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133(+) HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b(+) counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation. CONCLUSIONS: Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors.

Evaluation on the effect of different in-gel peptide isoelectric focusing parameters in global proteomic profiling.

Peptide isoelectric focusing (IEF) is a common technique used in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) proteomic workflow, in which the tryptic peptide is first pre-fractionated based on pI values before being subjected to reverse phase LC-MS analysis. Although this method has been widely used by many research groups, a systemic study on the optimal conditions and fundamental parameters influencing the experimental outcomes has been lacking, including the effect of peptide extraction methods, the extent of pre-fractionation, and the choice of pH range. In this study, we compared the effect of different parameters on the numbers of peptides and proteins identified using two complex mouse proteomes. The results indicated that extraction of peptides from immobilized pH gradient (IPG) strips by sequential elution of increasingly organic solvents provided the highest number of peptide identification. In addition, we showed that approximately 45 more unique proteins were identified for every additional fraction collected during peptide IEF. Although narrow pH ranges provided higher resolution in peptide separation as expected, different pH ranges yielded similar numbers of peptide and protein identification. Overall, we demonstrated that the extraction solvent influenced the numbers of peptide and protein identification and quantitatively demonstrated the advantage of extensive fractionation and the performance of different pH ranges in practice.

Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis.

Electroporation of cells with short, high-voltage pulses causes a transient permeabilization of cell membranes that permits passage of otherwise nonpermeating ions and molecules. In this study, we illustrate how electroporation with isotonic calcium can achieve highly effective cancer cell kill in vivo. Calcium electroporation elicited dramatic antitumor responses in which 89% of treated tumors were eliminated. Histologic analyses indicated complete tumor necrosis. Mechanistically, calcium electroporation caused acute ATP depletion likely due to a combination of increased cellular use of ATP, decreased production of ATP due to effects on the mitochondria, as well as loss of ATP through the permeabilized cell membrane. Taken together, our findings offer a preclinical proof of concept for the use of electroporation to load cancer cells with calcium as an efficient anticancer treatment. Electroporation equipment is already used clinically to enhance the delivery of chemotherapy to superficial tumors, with trials on internal tumors in progress, enabling the introduction of calcium electroporation to clinical use. Moreover, the safety profile, availability, and low cost of calcium facilitate access to this technology for many cancer patients in developed and developing countries.

Chemical characterization of a procyanidin-rich extract from sorghum bran and its effect on oxidative stress and tumor inhibition in vivo.

The present study was to characterize a procyanidin-rich extract (PARE) from sorghum ( Sorghum bicolor (L.) Moench) bran and assess its biological activities. The procyanidin oligomers were separated and identified by normal-phase HPLC equipped with fluorescence (FLD) and mass spectrometry (MS) detectors. In addition, the effects of PARE on oxidative stress in mice induced by D-galactose as well as tumor inhibition in C57BL/6J mice bearing Lewis lung cancer were investigated. Administration of D-galactose significantly (p < 0.05) lowered the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). This was accompanied by a significant (p < 0.05) increase in malondialdehyde (MDA) levels in both liver and serum. Administration of PARE (150 mg/kg) significantly (p < 0.05) reversed the d-galactose-induced oxidative stress by enhancing the activities of antioxidant enzymes. Furthermore, PARE administration inhibited tumor growth and metastasis formation by suppressing vascular endothelial growth factor (VEGF) production. The results suggested that PARE had antioxidant and antitumor activities.

Combination of human plasminogen kringle 5 with ionizing radiation significantly enhances the efficacy of antitumor effect.

Antiangiogenic therapy could destroy tumor vasculature and inhibit tumor growth. It might inhibit tumor growth significantly when used as a single treatment modality and its therapeutic benefit may even be greater when used in combination with established treatment modalities such as radiation therapy (RT). In the present report, we investigated the effect of recombinant human plasminogen kringle 5 domain (rhK5) in combination with ionizing radiation on angiogenesis, tumor growth and survival in a murine Lewis lung carcinoma (LLC) tumor model. Combined treatment using rhK5 and radiotherapy displayed obvious suppressive effect on LLC tumor growth as compared with single treatment with either modality (p < 0.05), and resulted in a more additive effect on tumor growth delay in this model. In addition, combined treatment significantly enhanced the survival of mice and no toxic effect, such as weight loss, was observed. The significant antitumor effect of rhK5 plus radiation was associated with a direct suppression effect on early neoangiogenesis and tumor cell apoptosis. Furthermore, the expression of VEGF and HIF-1alpha in tumor tissue correlated well with decreased vessel density. The results suggest that rhK5 significantly enhances the antitumor activity of RT and could be a potent adjuvant therapeutic approach to improve the efficacy of radiotherapy for lung cancer.

Tyroservatide therapy for tumor growth, invasion and metastasis of Lewis lung carcinoma and human lung carcinoma A549.

OBJECTIVES: The tripeptide tyroservatide (tyrosyl-seryl-valine, p-Tyr-Ser-Val-NH(2), YSV) has been shown to have anti-tumor effects on experimental hepatocarcinoma. The study was conducted to evaluate the therapeutic effects of YSV on tumor growth, invasion and metastasis of lung cancers. METHODS: Anti-tumor and anti-metastatic effects of YSV were evaluated in three experimental systems. In C57BL/6 mice, a spontaneous metastasis model of Lewis lung cancer was used to study the anti-tumor and anti-metastasis effects of YSV. A549 human lung carcinoma was used to create an orthotopic model in nude mice to investigate the anti-metastasis effect of YSV. Finally, an in vitro model using the B16F10 melanoma cell line was selected to observe the effect of YSV on adhesion and invasion, and to use immunocytochemistry to assay the expression of ICAM-1. RESULTS: YSV inhibited subcutaneous tumor growth of Lewis lung carcinoma (p < 0.05) and markedly decreased lung metastases in the spontaneous metastasis model of Lewis lung cancer and the orthotopic model of A549 human lung carcinoma. In vitro, YSV reduced adhesion and invasion as well as the expression of ICAM-1 in tumor cells. CONCLUSION: YSV was able to inhibit tumor growth and metastasis in lung cancer.

The role of copper in development of drug resistance in murine carcinoma.

Multidrug resistance (MDR) is a major obstacle to successful application of cancer chemotherapy and also a basic problem in cancer biology. Studies on the molecular basis of MDR have revealed that a number of proteins over express in multidrug resistant cells viz., multidrug resistant MDR1 gene product P-glycoprotein, the multidrug resistance-associated protein (MRP) and enzymes associated with the glutathione (GSH) metabolism. Decreased expression or altered activity of topoisomerase II has also been implicated in MDR. In the present investigation a number of changes in phase II detoxification parameters have been noticed in drug resistant cells but the novel aspect of the present report is the observation that the metal copper is involved in drug resistance. Although copper plays important roles in many human and other biological systems and even in the treatment of cancer but the relation of Cu and drug resistance has not so far been studied in detailed. The present report describes the novel findings that the level of copper increases with the development of drug resistance in Ehrlich ascites carcinoma and in Lewis lung carcinoma cells and also in serum of mice bearing drug resistant cancer cells compared to mice bearing drug sensitive cells; the work indicates the important aspect of treating drug resistant cancer patients by lowering Cu level in the cancerous cells and serum prior to treatment.

Tumor-derived prostaglandin E2 and transforming growth factor-beta stimulate endothelial cell motility through inhibition of protein phosphatase-2A and involvement of PTEN and phosphatidylinositide 3-kinase.

Tumor vascularization is a complex process that requires structural reorganization and increased motility by endothelial cells. Studies were conducted to identify the tumor-derived mediators and signaling pathways that lead to this increased endothelial cell motility. Using the Lewis lung carcinoma (LLC) tumor model, these studies showed that prostaglandin E2 (PGE2) and transforming growth factor-beta (TGFbeta) were the mediators that were responsible for the migration-stimulatory activity produced by the tumor cells. The response of endothelial cells to these tumor-derived motility-stimulatory factors involved a decline in the activity of the serine/threonine phosphatase PP-2A. Inhibition PP-2A either pharmacologically or genetically increased endothelial cell migration. Concurrent with the decline in PP-2A activity as a result of exposure to PGE2/TGFbeta was a loss of PP-2A co-precipitation with the inositol phosphatase PTEN and an increase in the PTEN serine phosphorylation level. Since hyperphosphorylation has been shown to inhibit the ability of PTEN to act as an antagonist to phosphatidylinositide 3-kinase (PI3K), the role of PI3K in PGE2/TGFbeta-stimulated migration was examined. These studies showed that the increased endothelial cell motility that resulted from PGE2/TGFbeta inhibition of PP-2A was dependent on PI3K.

[Significance of gefitinib combined with immunotherapy in tumor-bearing mice].

Gefitinib (brand name: Iressa) is a drug approved for molecule-targeting therapy in lung carcinoma patients. There are still many unresolved problems concerning the safety and mechanism of action of this drug. Based on the expectation that this drug combined with immunotherapy would be highly effective, we recently conducted an experiment in tumor-bearing mice. In this experiment, however, the tumor was not significantly reduced in size by this combined therapy. However, since the tumor in this animal model was EGFR positive and because tumor growth tended to be suppressed in mice with higher immune activity, it seems likely that additional studies of this therapy will contribute to identifying the optimum drugs to be combined with gefitinib and the optimum method of dosing that will lead to satisfactory efficacy and safety of gefitinib combined with immunotherapy.

Determinants of efficacy of immunotherapy with tumor-derived heat shock protein gp96.

Immunotherapy with gp96 was highly effective in mice bearing methylcholanthrene-induced fibrosarcomas (Meth A tumors) when treatment began 7 days or less after tumor challenge, but significantly less effective if the treatment began 9 days after challenge. Immunotherapy of pre-existing tumors showed all the hallmarks of specificity of gp96 and dose-restriction observed previously with prophylactic studies. When mice with large primary Meth A tumors were treated with surgery alone, or with surgery followed by therapy with Meth A-derived gp96, the mice that received surgery and immunotherapy did significantly better than those receiving surgery alone. The relationship between the time of initiation of immunotherapy with gp96 and its efficacy was also tested in a metastatic model of the Lewis lung carcinoma. In this model, immunotherapy with gp96 was very effective if treatment began up to 31 days after tumor challenge, but significantly less so if therapy was initiated day 33 post-tumor challenge. These observations suggest that the regulatory phenomena that interfere with immunotherapy gather momentum with surprising speed.

Screening of N-acylneuraminic acids in serum and tissue specimens of mouse C57BI with Lewis' lung cancer by high-performance liquid chromatography.

Serum and tissue specimens from healthy C57BI mice and from mice with Lewis' lung cancer after metastasis were analyzed for N-acetyl- and N-glycolylneuraminic acid by high-performance liquid chromatography. Both neuraminic acids were present, while N-glycolylneuraminic acid was the predominant sialic acid in all tissues. Samples from mice with metastatic cancer showed a significant increase (67-200%) of total sialic acids mainly as a result of increased N-glycolylneuraminic acid synthesis. These results suggest that cancer metastasis in various tissues is closely associated with increased synthesis of the predominant neuraminic acid and may help to understand the underlying mechanisms of tumor development.

Construction of a quantitative PCR system to determine expression of tumor associated antigen.

Cancer immunotherapy is a strategy for cancer treatment by induction of anti tumor responses. The identification of candidate tumor associated antigens (TAA) suggested their potential use as immunogens for vaccination studies. Quantification of a TAA expression by cancerous cells is an important factor in determination of induced immune response efficiency against tumors and thus enables us to devise optimal immunotherapy protocol to cure cancer. The quantitative polymerase chain reaction (PCR) enables us to compare the TAA expression in highly metastatic tumor clones with that in less metastatic ones and in normal cells. It allows us to gain more insight into genome rearrangements that occur in malignant transformation as well as broaden our knowledge about tumor cell gene expression regulation. One of the peptides isolated previously in our lab from a murine lung carcinoma is the mutated Connexin 37 (cx37), a gap-junction protein. Research is underway to determine the expression level of the TAA in various Lewis lung carcinoma cells. This evaluation is achieved by means of quantitative PCR. A quantitative PCR experiment includes preparation of a control template, which is added in known amounts together with the target template in a series of amplification reactions. The control template uses the same primers as the target sequence, yet their PCR products differ in size so as to be distinguishable. Two methods were used to produce this control template. The first one included specific deletion of a sequence of approximately 100 bp that lay between the two primers, insertion of the new template into a plasmid vector, transformation of competent bacteria, detection of transformed bacterial colonies and isolation of the plasmid DNA in a large quantity. The non-mutated, deleted Connexin 37 cDNA was also isolated from bacteria and used for another experiment aimed at producing deleted, mutated Connexin 37 cDNA by means of primer mutagenesis. (Fig. 5, Ref 8.)

Influence of lipid diets on the number of metastases and ganglioside content of H59 variant tumors.

We investigated the influence of the fatty acid composition of the diet on the number of hepatic metastases and the ganglioside profile of the primary tumor and metastases. C57BL/6 female mice were fed different diets containing either no fats (TEK) or 8% of fish oil (POL), linseed oil (LIN), safflower oil (SAF) or beef tallow (BT) and were injected subcutaneously in the dorsum with H59 cells, a variant of the Lewis lung carcinoma (3LLc) that metastasizes preferentially to the liver. The omega3 polyunsaturated fatty acid (PUFA)-rich diets (LIN and POL) elicited more metastases than the omega6 PUFA-rich (SAF), fat-free (TEK), or saturated fats (BT) diets. However, dietary fat did not influence the ganglioside composition of either the primary tumors or the metastases, at least in the glucidic part. However, comparison of diets with low (TEK, SAF, and BT) and high (LIN and POL) number of metastases showed that the levels of G3 (which could be a second band of GM2) were greater in metastases of the latter group. This study showed that the H59 hepatic metastases contained more GM2 than the s.c. tumors, irrespective of diet or the number of metastases produced. The small differences in the ganglioside profiles observed in this study could have resulted from the limitations of the HPTLC method. A detailed analysis of the lipid chains, as well as glycolipids other than gangliosides, could give more information on changes resulting from different lipid diets.