Sanjaya K. Mallick

Antiproliferative and immunostimulatory protein fraction from edible mushrooms.

Fruit bodies and mycelia of various higher Basidiomycetes were studied in search of biological effector molecules. In this study, we evaluated the antiproliferative and immunomodulatory properties of a protein fraction designated as Cibacron blue affinity eluted protein (CBAEP) isolated from five different species of edible mushrooms (Termitomyces clypeatus, Pleurotus florida, Calocybe indica, Astraeus hygrometricus, and Volvariella volvacea). This protein fraction (10-100μg/ml) mediated antiproliferative activity on several tumor cell lines through the induction of apoptosis. Also the isolated protein fraction from all five mushrooms had a stimulatory effect on splenocytes, thymocytes and bone marrow cells. Further it enhanced mouse natural killer (NK) cell cytotoxicity and stimulated macrophages to produce nitric oxide (NO). The highest immunostimulatory activity was determined in the CBAEP from T. clypeatus and the highest antiproliferative activity from C. indica.

In vitro immunostimulatory properties of Abrus lectins derived peptides in tumor bearing mice

In vitro immunostimulatory effect of Abrus lectins derived peptide fractions (AGP and ABP) was investigated in DL bearing mice. Both AGP and ABP were found to activate splenocytes and induced production of cytokines like IL-2, IFN-gamma and TNF-alpha indicating a Th1 type of immune response. Analysis of in vitro treated splenocytes by flow cytometry revealed an increase in percentage of T and B cell with high expression of activation markers (CD25(+) and CD71(+)). At the same time, expression of co-stimulatory markers was significantly high compared to tumor control. The tumor associated macrophages were able to stimulate NO production, IL-1 secretion, increased phagocytosis and decreased expression of mannose receptor. It was also observed that NK cell was activated by AGP and ABP. These results suggest that both AGP and ABP act as immunostimulants in vitro in DL bearing mice.

Antitumor and proapoptotic effect of Abrus agglutinin derived peptide in Dalton's lymphoma tumor model.

Abrus agglutinin peptide fractions obtained from 10 kD molecular weight cut off membrane permeate (10 kMP), was shown to have selective antiproliferative activity on several tumor cell lines with induction of apoptosis through mitochondrial pathway. The present study was designed to evaluate acute general toxicity and in vivo therapeutic effectiveness of 10 kMPP in Daltons lymphoma (DL) mice model. The acute toxicity like body weight, peripheral blood cell count, lympho-hematological and biochemical parameters remained unaffected with 1mg/kg body weight and lower of 10 kMPP. The in vivo antitumor study indicated that there were 27%, 58.5% and 84.5% reduction in DL cell survival in 100, 200 and 500 microg/kg body weight of 10 kMPP, respectively. Analysis of the growth inhibitory mechanism in DL cells revealed nuclear fragmentation and condensation with appearance of the sub G0/G1 peak is indicative of apoptosis. Further, the Western blotting showed apoptosis was mediated by reduction in ratio of Bcl-2 and Bax protein expression, and activation of caspase-3 through release of cytochrome-c in DL cells. Kaplan-Meier survival analysis showed an effective antitumor response (53 ILS%) with dose of 500 microg/kg body weight. Our result showed that the novel peptides present in Abrus agglutinin possess potent antitumor properties which need to be further explored.

Tumour escape mechanisms and their therapeutic implications in combination tumour therapy

Most tumours arise from a single normal cell through a sequential evolutionary process of mutation and selection. Tumours are initiated by escaping non‐immune surveillance, which includes defective DNA repair, epigenetic gene alternation, resistance to apoptosis and loss of intercellular contact inhibition. Tumour cells harbour mutations in a number of critical genes that provide selective advantages at various stages during the evolution of the tumour. The tumour cells that circumvent the tumour suppressor mechanisms of the non‐immune surveillance process are edited by the immune system, resulting in the selection of a resistant tumour variant. The selection of the tumour cell is further shaped by its interactions with cells and other factors in its microenvironment. Tumour evolution is thought to adhere to Darwinian principles by escaping both non‐immune (intrinsic) and immune (extrinsic) responses against self‐altered tumour cells. At end‐stage, tumours have escaped both non‐immune and immune surveillance with increased threshold of apoptosis. Combination therapy has been proposed, by exploring the non‐immune and immune suppressive nature of the tumour, and has been found to have a therapeutic efficiency on tumour regression as compared with monotherapies. The combination of immunotherapy and other different modalities, especially vaccines, with conventional anticancer therapies with optimized dosage and scheduling can offer synergistic antitumour effects. Here, we focus on the mechanism of tumour evolution and its implication in combination therapy.

Abrus abrin derived peptides induce apoptosis by targeting mitochondria in HeLa cells

In our previous study, Abrus abrin derived peptide fraction (ABP) with molecular weight in range of 600–1500 Da was shown to have potent antitumor activity in Daltons lymphoma (DL) tumor bearing mice. The purpose of this study was to elucidate the mechanism of mitochondrial apoptosis induced by the peptide fraction. ABP was found to have selective antiproliferative activity (10 ng–100 ng/ml) on several tumor cell lines in vitro without having any cytotoxic effect on normal cell lines with a dose of 1000 ng/ml. Analysis of the growth inhibitory mechanism in HeLa cells revealed DNA fragmentation with appearance of the sub G0/G1 peak indicative of apoptosis. Further investigation results showed that the apoptotic machinery of HeLa induced by ABP was associated with the release of reactive oxygen species, a drop in mitochondrial transmembrane potential, upregulation of Bax, downregulation of Bcl‐2, and activation of caspase‐3. The peptide fraction was found to target mitochondria of HeLa cells as observed by confocal microscopy. This peptide fraction offers a source of mitochondria penetrating peptides which might have therapeutic induction of apoptosis in cancer cells.

Immunostimulatory Properties of a Polysaccharide Isolated from Astraeus hygrometricus

Mushrooms have been used as food and for therapeutic purpose for decades, and various compounds derived from these have potential biological activities. Application of such agents of natural origin is vital with relevance to an increase in diseases involving immune dysfunction, cancer, and autoimmune conditions in recent years. These compounds belong mainly to polysaccharides, especially beta-D-glucan, glycopeptide/protein complexes, proteoglycans, proteins, and triterpenoids. Among these compounds, polysaccharides and their peptide/protein derivates have extremely important roles in immunomodulating activities. The present study aims to explore the immunomodulatory properties of polysaccharide isolated from the mushroom Astraeus hygrometricus. The polysaccharide isolated by hot alkaline extraction and chromatography was designated as AE2 and studied for its immunostimulatory potential in vivo in a murine model. Macrophages from treated mice showed higher production of nitric oxide and interleukin-1 and also exhibited an increased phagocytic potential. It also enhanced natural killer cell activation and proliferation of splenocytes with an increase in the T-helper (Th) 1 cytokine level of splenocyte culture supernatant. The cytokine level in serum also indicated a Th1 skew. The observed activities were associated with no general toxicity to the organism. The findings will be helpful in considering A. hygrometricus as a potential source of an immunomodulator and in designing further studies to understand its mode of action on immune system.

Activation of RAW 264.7 cells by Astraeus hygrometricus- derived heteroglucan through MAP kinase pathway

Mushroom‐derived polysaccharides like β‐glucan are being investigated for therapeutic properties for a long time, but their mode of action of immunomodulatory properties is not well established. In the present study, a heteroglucan from Astraeus hygrometricus designated as AE2 is investigated for its macrophage stimulatory properties using RAW 264.7 cell line. An augmentation of nitric oxide production is observed in the presence of AE2 in a dose‐dependent manner due to up‐regulation of iNOS (inducible NO synthase) expression; hence NF κB (nuclear factor κB) pathway is investigated. RAW 264.7 cells endured phosphorylation of Ikk (IκB kinase) and subsequently NF κB is translocated to the nucleus. Further, the PKC (protein kinase C) level of the cells enhanced significantly. We also found that AE2 could induce the phosphorylation of p38 MAPK (mitogen‐activated protein kinase), ERK1/2 (extracellular‐signal‐regulated kinase 1/2), MEK (MAPK/ERK kinase) and JNK (c‐Jun N‐terminal kinase), whereas it failed to induce phosphorylation of JAK2 (Janus kinase 2) and STAT1. These results indicated that the macrophage activation by AE2 might be exerted, at least in part, via MAPKs (mitogen‐activated protein kinases) pathway of signal transduction.

Antitumor properties of a heteroglucan isolated from Astraeus hygrometricus on Dalton’s lymphoma bearing mouse

Polysaccharides such as glucans are the best known and most potent mushroom-derived substances with antitumor and immunomodulating properties. However, a vast variety of mushroom species remain unexplored. The present study aims at exploring the possible role of a heteroglucan designated as AE2, isolated from Astraeus hygrometricus, on tumor bearing host. AE2 mediated tumor regression through the reversal of tumor induced immunosupression. Tumor-associated macrophages from AE2 administered tumor bearing host have been demonstrated to possess augmented surface expression of co-stimulatory molecules and MHC-II, representing essentially elevated state of macrophage activation. NK cell activity has also been found to be elevated with the AE2 treatment while the Th1 cytokine in the tumor micro-environment increases with the treatment, the Th2 cytokine levels concomitantly decreases. Further, following the immunoactivation, Daltons lymphoma (DL) cells has undergone apoptosis, thereby resulting in reduction of tumor growth and increased survival rate of the tumor bearing host. The present study sheds light on the potent antitumor property of the heteroglucan AE2 isolated from A. hygrometricus, and can be extended further to develop therapeutic protocols for treatment of cancer or disease resulting in immunosuppression.

Inhibitory effect of Abrus abrin-derived peptide fraction against Dalton's lymphoma ascites model

Peptides derived from larger molecules that are important modulators in cancer regression are becoming leads for development of therapeutic drugs. It has been reported that Abrus abrin, isolated from the seeds of Abrus precatorius, showed in vitro and in vivo antitumor properties by the induction of apoptosis. The present study was designed to evaluate the in vivo therapeutic effectiveness of abrin-derived peptide (ABP) fraction in Daltons lymphoma (DL) mice model. The lethal dose (LD(50)) of ABP was found to be 2.25 mg/kg body weight and further the acute toxicity was determined with sublethal doses in normal mice. The acute toxicity like body weight, peripheral blood cell count, lympho-hematological and biochemical parameters remained unaffected till 200 microg/kg body weight of ABP. The sublethal doses of ABP showed very significant growth inhibitory properties in vivo DL mice model. There were 24%, 70.8% and 89.7% reductions in DL cell survival in 25, 50 and 100 microg/kg body weight of ABP, respectively. Analysis of the growth inhibitory mechanism in DL cells revealed nuclear fragmentation, and condensation with the appearance of the sub-G(0)/G(1) peak is indicative of apoptosis. Further, the Western blotting showed that apoptosis was mediated by the reduction in the ratio of Bcl-2 and Bax protein expression, and activation of caspase-3 through the release of cytochrome c in DL cells. Kaplan-Meier survival analysis showed an effective antitumor response (104.6 increase in life span (ILS) %) with a dose of 100 microg/kg body weight.

Stimulation of murine B and T lymphocytes by native and heat-denatured Abrus agglutinin

In our previous studies, Abrus agglutinin showed non-specific immunostimulatory activity both native (NA) and heat-denatured (HDA) condition in mouse model. The present study was investigated to decipher the specific immune response towards B and T cell by NA and HDA. It was observed that the proliferation index for NA and HDA of stimulated B cells are 1.35 and 1.41 respectively and on the other hand, T cell proliferation index for NA and HDA are 1.67 and 1.54 respectively. At the same time, expression of surface and activation marker for B and T cells was significantly different compared to control as quantified by flow cytometry. But the expression of co-stimulatory markers (CD 80 and CD 86) was not significantly different and NA and HDA in immunized splenocytes with Daltons lymphoma antigen induced antibodies titer 4.37 and 4.2 times more than control. This study indicates Abrus agglutinin (NA and HDA) acts as a B cell and T cell stimulator.