Pramod Kumar Gautam

A benzophenanthridine alkaloid, chelerythrine induces apoptosis in vitro in a Dalton's lymphoma

PURPOSE The aim of this study was to investigate the effect of chelerythrine on DL cell apoptosis in an in vitro experimental setup. MATERIALS AND METHODS For tumor model, spontaneous occurring T-cell lymphoma designated as Daltons lymphoma (DL) was selected. Double staining, transmission electron microscope (TEM), fluorescence microscopy, Western blotting, Reverse Transcriptase-Polymerase Chain Reaction, and DNA fragmentation assay were used to detect heat shock factor 1 (HSF1) and hsp70 expression and PKC phosphorylation, and apoptotic characteristic of DL cells. RESULTS Chelerythrine exposure resulted in significant morphological alteration comparable to that of apoptosis. Furthermore, it was confirmed by fluorescence microscopy, TEM analysis, and DNA fragmentation assay that 10 μg/mL of chelerythrine is capable of inducing apoptosis in DL cells. The suppression in HSF1 expression and subsequent inhibition of hsp70 expression in chelerythrine-treated DL cells suggest that chelerythrine induces apoptosis in DL cells by inhibiting the expression of these cytoprotective proteins. CONCLUSION Chelerythrine is capable of inducing apoptosis DL cells in vitro and therefore, it could be useful in combating tumor growth and progression.

Progressive growth of a murine T cell lymphoma alters population kinetics and cell viability of macrophages in a tumor-bearing host

Tumor progression induces infiltration of immune cell populations at the site of tumor growth. Infiltrated leukocyte population including monocyte and macrophages interacts with tumor cells and tumor microenvironment and results in the suppression of macrophage functions. Impaired functions of macrophages result in the suppression/inhibition of cell-mediated immunity leading to inefficient antitumor immune responses. Impaired macrophage population invariably helps in immune selection of tumor leading to uninterrupted growth and progression in the host. Murine T cell lymphoma designated as Dalton’s lymphoma is highly immunosuppressive and invasive tumor of T cell origin, which completely paralyzes the host’s immune system resulting in a very short life span of the host. Progressive growth of Dalton’s lymphoma (DL) cells has been known to inhibit the release of inflammatory cytokines and effector mediator molecules. In this study, we demonstrate that intraperitoneal transplant of DL cells in normal healthy host induces a rapid increase in macrophage cell population during early stage of tumor progression and progressive decrease in tumor-associated macrophage population and reduced survival of macrophages in advance stage of tumor burden.

Selenium nanoparticles induce suppressed function of tumor associated macrophages and inhibit Dalton's lymphoma proliferation

Selenium Nanoparticle (SeNPs) is reported that it enhances and maintains optimal immune during infection and malignancies. To this end, we examined the role of selenium on TAMS whose anti-tumor function suppressed which favor tumor progression. BALB/c (H2d) strain of mice non-Hodgkin type of Daltons cell line was used to check the role of carboxlic group induced, synthesized SeNPs on TAMs. Screening of IC50 value was done primarily trypen blue exclusion assay and 50% proliferation of DL cells inhibited 40 ng/ml to 50 ng/. Treatment also decreases ΔΨm, fragmentation of DNA of DL cells and arrest cells cycle in G1/G0 phage. Untreated TAMs cells showing suppressed expression of ROS, adhesion, phagocytosis, fusion and receptor profiling such as ICAM-1, CD47, CD172α. Which was induced more as compare to untreated group. SeNPs have potential to induce the anti-tumor function of TAMs whose anti-tumor function down-regulated pliable shifted towards tumor progression. It decreased the proliferation of DL cell by inducing apoptosis. Therefore, the synthesized SeNPs could be used for imaging diagnosis and cancer therapy which must be cost effective with negligible side effects shifted towards tumor progression. It decreased the proliferation of DL cell by inducing apoptosis.

Antigenic Hsp70–peptide upregulate altered cell surface MHC class I expression in TAMs and increases anti-tumor function in Dalton’s lymphoma bearing mice

Major histocompatibility complex (MHC) class I molecules not only provide a mechanistic framework for the cell-to-cell communication, but also possess broader biological function. Due to their ability to regulate presentation of tumor-associated antigens (TAAs), viral peptides which play an essential role in the regulation of immune responses by presenting antigenic peptides to cytotoxic T lymphocytes and by regulating cytolytic activities of immune cells. Tumor cells frequently do not express MHC class I molecules; as a result, tumor cells escape from immune surveillance. Cells surviving in tumor microenvironment are often characterized by a profound immune escape phenotype with alterations in MHC class I way of antigen processing. Cellular components of the tumor microenvironment, in particular alternatively activated M2 phenotype, are involved in tumor progression and suppression of anti-tumor immunity. Hsp70 is well recognized for its role in activating macrophages leading to enhanced production of inflammatory cytokines. It has been observed that Hsp70 derived from normal tissues do not elicit tumor immunity, while Hsp70 preparation from tumor cell associated with antigen are able to elicit tumor immunity. The finding shows that the expression of MHC class I (H2Db) drastically decreases in TAMs and Hsp70–peptide complex enhances H2Db expression in TAMs and it reverts back the suppressed function of TAMs into the M1 state of immunoregulatory phenotype that promotes tumor regression by enhanced antigen presentation.

Role of Macrophage in Tumor Microenvironment: Prospect in Cancer Immunotherapy

Current evidence suggests an increasing role of macrophages in inflammation and tumor progression. Most tumors contain an abundant number of macrophages as a major component of their leukocyte infiltrate, which co-exist with tumor cells at the tumor microenvironment. Upon activation with soluble tumor antigens, macrophages release a distinct repertoire of growth factor, cytokines, chemokines and enzymes that inhibit growth of the tumor. However, the anti-tumor immune response induced by macrophages does not always ensue. Tumor cells themselves are capable of down-regulating macrophage phenotype and functions and anti-tumor immune responses in the tumor-bearing host. The present review aims to elucidate the role of macrophages in tumor growth and progression, invasion, metastasis, and angiogenesis at the site of tumor growth. Moreover, the effect of tumor microenvironment on the phenotype and function of macrophages, which are altered due to the continuous exposure of various soluble and non-soluble tumor promoting factors secreted by tumor cells, and implication of macrophages in cancer immunotherapy have been discussed in detail.

NK Cell Effector Functions Regulation by Modulating nTreg Cell Population During Progressive Growth of Dalton’s Lymphoma in Mice

ABSTRACT Natural killer (NK) cells are large granular lymphocytes of the innate immune system and play a pivotal role against virus-infected cells, microbial pathogens, and tumor cells. NK cells secrete several cytokine,s but IFN-γ secreted by NK cells play a vital role in the activation of the innate and adaptive immune systems. But during any infection or tumor burden, functional activity of NK cells is downregulated significantly by nTreg cells. It is also found that during tumor progression, the number of nTreg cells increases as a result; it effectively suppresses the antitumor activity of NK cells. Therefore, in the present investigation, we intend to examine the mechanism of downregulation of antitumor immune response mediated by NK cells. We observed increased NK cell population at an early stage of Dalton’s lymphoma (DL) growth, while at late stage, NK cell numbers were decreased. The NK cell functional activity was govern by high level of IFN-γ measurement during tumor progression. The FoxP3+ CD25+ CD4+ T regulatory cell population was found to be continuously increased with high-level expression of FoxP3 during DL growth. The rapid increase in the number of Treg cells during DL progression may be due to high level of the FoxP3 transcription factor. The tumor microenvironment of DL cell progression has highly deleterious effect on NK cells after massive growth of tumor burden in BALB/c mice. This result also indicates that NK cell proliferation, activation, and accumulation are under the control of regulatory T cells.

Biologically Synthesized Gold Nanoparticles using Ocimum sanctum (Tulsi Leaf Extract) Induced Anti-Tumor Response in a T Cell Daltons Lymphoma

The characterizations of green synthesized gold nanoparticles (AuNPs) prepared using Ocimum sanctum leaf extract were done by UV-Spectrophotometer at 500-540 nm. The XRD data obtained were found similar to gold JCPDS File No- 04-0784. SEM and TEM analysis of AuNPs revealed spherical shape and size of 200 nm. Further FT-IR data indicated the various biomolecules present in Ocimum sanctum leaf extract provides stability to gold nanoparticles synthesis. The AuNPs were studied for their anti-cancer activity on Dalton’s lymphoma (DL) cells and the results obtained with IC50 value of < 50 ng/ml performed by MTT assay. Further, to confirm anti-tumor potential and the mode of action of the synthesized AuNPs, cell viability assay, nuclear morphology, DNA fragmentation assay, mitochondrial membrane potential (ΔΨm) analysis, and cell cycle analysis were done using DL cells. DL cells treated with the AuNPs showed reduced cell viability, altered nuclear morphology, typical apoptotic DNA ladder formation and apoptosis. From the above finding it can be concluded that the AuNPs have potential to decrease the proliferation of tumor cells and enhanced the production of ROS. Gold nanoparticles used in cancer detection and diagnosis/treatment are mainly in preclinical stages of cancer development.

Protein kinase C-α and the regulation of diverse cell responses

Abstract Protein kinase C (PKC) comprises a family of lipid-sensitive enzymes that have been involved in a broad range of cellular functions. PKC-α is a member of classical PKC with ubiquitous expression and different cellular localization. This unique PKC isoform is activated by various signals which evoke lipid hydrolysis, after activation it interacts with various adapter proteins and is localized to specific cellular compartments where it is devised to work. The universal expression and activation by various stimuli make it a perfect player in uncountable cellular functions including differentiation, proliferation, apoptosis, cellular transformation, motility, adhesion and so on. However, these functions are not intrinsic properties of PKC-α, but depend on cell types and conditions. The activities of PKC-α are managed by the various pharmacological activators/inhibitors and antisense oligonucleotides. The aim of this review is to elaborate the structural feature, and provide an insight into the mechanism of PKC-α activation and regulation of its key biological functions in different cellular compartments to develop an effective pharmacological approach to regulate the PKC-α signal array.

CD28-mediated T cell response is upregulated by exogenous application of autologous Hsp70–peptide complex in a tumor-bearing host

Abstract Hsp70, a highly conserved protein, has gained plenty of attention by virtue of its adjuvant capability to induce peptide-specific cytotoxic T lymphocyte responses. In this study, we have investigated the effect of autologous Hsp70–peptide complex (or simply autologous Hsp70) on the expression of CD28 on T cells and its effector functions through macrophage activation. Further, we investigated the effect of Hsp70 on the expression of CD80 and CD86 on macrophages isolated from normal and tumor-bearing host to provide costimulatory signal for T cell activation and secretion of IL-2 and IFN-γ during interaction. We found that treatment of autologous Hsp70 effectively activated TAMs to induce higher expression of CD28 on T cells through T cells–macrophage interaction. Treatment of autologous Hsp70 induces higher expression of CD80 and CD86 on TAMs, as a result, increases B7/CD28 interaction, which in turns activates T cells and induces higher production of IL-2 and IFN-γ, thereby increasing antigen-specific T cell proliferation. With our novel study, we have provided the strong insights into the role of extracellular Hsp70 on the expression of CD28 costimulatory molecule on T cells, which helps in the activation and generation of antigen-specific T cell effector functions in a tumor-bearing host to curb malignancy.