Detection of Cancer Cell Death Mediated by a Synthetic Granzyme B-like Peptide Fluorescent Conjugate and the same Peptide Binding in Bacteria

Granzyme-mediated apoptosis, supported by pore-forming perforin, plays an important role in CD8+ T lymphocytes (CTL)-dependent cellular immunity protection against both cancer and viral infection. Quantitative and qualitative problems with CTL are potential contributing factors to disease progression. The feasibility of developing CTL-independent cellular immunity is desired but must first overcome the barrier of CTL-independent target cell recognition. Granzyme B with its strong pro-apoptotic activity in many different target cells is investigated for use in the CTL-independent cellular immunity approach, and granzyme B or its bioactive peptides without the enzymatic activity are more desirable for use. Native granzyme B with enzymatic activity is usually investigated in cancer cells for its mediation of apoptosis by detection of DNA fragmentation. Detection of cell death mediated by such peptides in cancer cells is needed to demonstrate the potential therapeutic purposes. We show with never-before-seen microscopic images using fluorescence microscopy that a synthetic granzyme B-like peptide fluorescent conjugate (GP1R) can: 1) mediate cell death of different cancer cells via membrane extrusion, 2) bind to constitutively expressed binding targets in different cancer cells and bacteria, and 3) promote bacterial phagocytosis. The putative binding targets may serve as a universal pathologic biomarker detectable by GP1R. Our data taken together demonstrate the potential applications of GP1R for use in CTL-independent target cell recognition and target cell death induction. It may lead to development of rapid targeted detection and new treatment of cancer, viral and bacterial infections. The new treatment may show mutual benefits for two or more diseases.     

Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λ _max) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.     

Detection of Binding of a Synthetic Granzyme B-like Peptide Fluorescent Conjugate within Platelet-like Structures in Cancer-related Peripheral Blood Specimens and Tissue Sections

Platelets and cytotoxic T lymphocytes (CTL) are important whole blood components in peripheral blood. Studies have shown that platelets, from precursor megakaryocytes, are significant factors in cancer prognosis, cancer progression, and metastasis; but a direct platelet-cancer relationship remains unclear. CTL play an essential role in cancer surveillance by inducing cancer cell death with granzyme B. A recent report has shown the presence of binding targets with binding affinity to a synthetic granzyme B-like peptide fluorescent conjugate (GP1R) in different types of cancer cells grown in vitro. It suggests that these binding targets may serve as a “universal-pathologic-biomarker”. It is not known if similar biomarkers may be present in platelets of cancer patients. We show with fluoroscopic images that GP1R can bind to binding targets: 1) within platelets in methanol-fixed whole blood smears of patients with breast cancer and lung cancer, and 2) within platelet-like structures in formalin-fixed-paraffin-embedded (FFPE) nude mouse xenogeneic breast tumor tissues. Samples without cancer-association displayed no discernible GP1R-binding in platelet-like structures. Our data demonstrate for the first time that a similar “universal-pathologic-biomarker” detectable by GP1R-binding is present in circulating platelets of cancer patients. The data depict a co-existence of animal-platelets and human-breast cancer cells, both have a common pathologic biomarker detectable by GP1R, in the tumor growth. The fluoroscopic images indicate a visual direct connection between pathologic platelets and cancer. These preliminary results may lead to developments of novel platelet-based cancer diagnostics and therapeutics and a better understanding of the potential multifunction of GP1R and its relationship to megakaryocytes and PD1.     

Connecting Effective Immune Response,Fluorescent Granzyme B-like Peptide,Specific Peptide Binding Patterns,Patients with Cancer and Viral Infection,in Remission,Clinical Significance,and Liquid Biopsy

Functional cytotoxic-T-lymphocytes (CTL) with granzyme B play an important role in an effective immune response to tumor growth and infection progression. Tumor cells and platelets in peripheral whole blood smears of cancer patients have shown the presence of innate binding targets for GP1R, a fluorescent synthetic Granzyme B-like peptide. It is not known if similar GP1R-binding targets and specific binding patterns are detectable in peripheral blood of patients with viral infection. It is also not known if a specific binding pattern may be associated with an effective immune response to indicate a favorable prognosis. We reviewed the GP1R-binding patterns in the peripheral blood smears of 5 patients in remission at the time of sampling (3 with cancer and 2 with flu-like symptoms) and a negative control. We show with fluoroscopic images that there are: 1) fluorescent GP1R-binding targets mostly in the cytoplasmic areas of nucleated cells in patients with breast and lung cancer who have longer survival, 2) intense fluorescent deposits mostly in the nuclear areas of segmented neutrophils in patients recovered from severe to mild flu-like symptoms, 3) discernible fluorescent deposits in the cytoplasmic areas of small lymphocyte-like elements and overall intense fluorescent stain in large cells in the patient with advanced pancreatic cancer who had shorter survival, 4) GP1R-binding targets in numerous platelet-like elements in all 5 patients. The control sample did not show similar binding patterns. The potential association between specific GP1R-binding patterns in peripheral blood samples and prognostic significance, and its use as liquid biopsy are discussed.