Exploring the immunomodulatory potential of Brahmi (Bacopa monnieri) in the treatment of invasive ductal carcinoma.

Bacopa monnieri (L) Wettst, commonly known as Brahmi, stands as a medicinal plant integral to India's traditional medical system, Ayurveda, where it is recognized as a "medhya rasayana"-a botanical entity believed to enhance intellect and mental clarity. Its significant role in numerous Ayurvedic formulations designed to address conditions such as anxiety, memory loss, impaired cognition, and diminished concentration underscores its prominence. Beyond its application in cognitive health, Brahmi has historically been employed in Ayurvedic practices for the treatment of inflammatory diseases, including arthritis. In contemporary biomedical research, Bacopa monnieri can attenuate the release of pro-inflammatory cytokines TNF-α and IL-6 in animal models. However, there remains a paucity of information regarding Bacopa's potential as an anticancer agent, warranting further investigation in this domain. Based on previous findings with Brahmi (Bacopa monnieri), the current study aims to find out the role of Brahmi plant preparation (BPP) in immunomodulatory actions on IDC. Employing a specific BPP concentration, we conducted a comprehensive study using MTT assay, ELISA, DNA methylation analysis, Western blotting, ChIP, and mRNA profiling to assess BPP's immunomodulatory properties. Our research finding showed the role of BPP in augmenting the action of T helper 1 (TH1) cells which secreted interferon-γ (IFN-γ) which in turn activated cytotoxic T-lymphocytes (CTL) to kill the cells of IDC (*p < 0.05). Moreover, we found out that treatment with BPP not only increased the activities of tumor-suppressor genes (p53 and BRCA1) but also decreased the activities of oncogenes (Notch1 and DNAPKcs) in IDC (*p < 0.05). BPP had an immense significance in controlling the epigenetic dysregulation in IDC through the downregulation of Histone demethylation & Histone deacetylation and upregulation of Histone methylation and Histone acetylation (*p < 0.05). Our Chromatin immunoprecipitation (ChIP)-qPCR data showed BPP treatment increased percentage enrichment of STAT1 & BRCA1 (*p < 0.05) and decreased percentage enrichment of STAT3, STAT5 & NF ΚB (*p < 0.05) on both TBX21 and BRCA1 gene loci in IDC. In addition, BPP treatment reduced the hypermethylation of the BRCA1-associated-DNA, which is believed to be a major factor in IDC (*p < 0.05). BPP not only escalates the secretion of type 1 specific cytokines but also escalates tumor suppression and harmonizes various epigenetic regulators and transcription factors associated with Signal Transducer and Activator of Transcription (STAT) to evoke tumor protective immunity in IDC.

Epigenetic regulation of enhancer of zeste homolog 2 (EZH2) -Yin Yang 1 (YY1) axis in cancer.

In accordance with the World Health Organization, cancer is the second leading cause of death in patients. In recent years, the number of cancer patients has been growing, and the occurrence of cancer in people is becoming more common, primarily due to lifestyle factors. Yin Yang 1 (YY1) is a transcription factor that is widespread throughout. It is a zinc finger protein, falling under the GLI-Kruppel class. YY1 is known to regulate transcriptional activation and repression of various genes associated with different cellular processes such as DNA repair, autophagy, cell survival and apoptosis, and cell division. Meanwhile, EZH2 is a histone-lysine N-methyltransferase enzyme encoded by gene 7 in humans. Its main function involves catalyzing the addition of methyl groups to histone H3 at lysine 27 (H3K27me3), and it is involved in regulating CD8 + T cell fate and function. It is a subunit of a Polycomb repressor complex 2 (PRC2). The EZH2 gene encodes for an enzyme that is involved in histone methylation and transcriptional repression. It adds methyl groups to lysine 27 on histone H3 (H3K27me3) with the help of the cofactor S-adenosyl-L-methionine. In addition to its role in epigenetic regulation, EZH2 also acts as a regulator of CD8+ T cell fate and function. EZH2 has been implicated in T Cell Receptor (TCR) signaling via the regulation of actin polymerization. In fact, EZH2 is involved in numerous signaling pathways that lead to tumorigenesis. EZH2 is mutated in cancer and shows overexpression. Due to its mutation and overexpression, the cells that help combat cancer are suppressed and carcinogenicity is promoted. The association of EZH2 and YY1 poses an intriguing mechanism in relation to cancer.

Depletion of enhancer zeste homolog 2 (EZH2) directs transcription factors associated with T cell differentiation through epigenetic regulation of Yin Yang 1(YY1) in combating non-small cell lung cancer (NSCLC).

Non-Small Cell Lung Cancer (NSCLC) is the leading cause of death in all countries alike. In the current study, we have found out that Histone H3Lys4trimethylation is abnormal on YY1 in CD4+T Helper (TH) cells of NSCLC patients which is evident by Histone H3Lys27 trimethylation mediated via EZH2. We investigated the status of Yin Yang 1 (YY1) and the involvement of certain transcription factors that lead to tumorigenesis after depleting endogenous EZH2 in vitro by CRISPR/Cas9 in the CD4+TH1-or-TH2-polarized cells isolated initially as CD4+TH0 cells from the PBMC of the control subjects and patients suffering from NSCLC. After depletion of endogenous EZH2, RT-qPCR based mRNA expression analysis showed that there was an increase in the expression of TH1 specific genes and a decrease in the expression of TH2 specific genes in NSCLC patients CD4+TH cells. We can conclude that this group of NSCLC patients may have the tendency at least in vitro to elucidate adaptive/protective immunity through the depletion of endogenous EZH2 along with the reduction in the expression of YY1. Moreover, depletion of EZH2 not only suppressed the CD4+CD25+FOXP3+Regulatory T cells (Treg) but also it aided the generation of CD8+Cytotoxic T Lymphocytes (CTL) which were involved in killing of the NSCLC cells. Thus the transcription factors involved in EZH2 mediated T cell differentiation linked to malignancies offers us an appealing avenue of targeted therapeutic intervention for NSCLC.

Diospyros malabarica fruit preparation mediates immunotherapeutic modulation and epigenetic regulation to evoke protection against non-small cell lung cancer (NSCLC).

ETHNOPHARMACOLOGICAL RELEVANCE: Diospyros malabarica is an ethnomedicinal plant with hypoglycaemic, anti-bacterial, and anti-cancer properties and it belongs to the Ebenaceae family which is well known for its medicinal uses since ancient times and application of its bark and unripened fruit has been significantly mentioned in Ayurvedic texts. The Diospyros malabarica species which is known as the Gaub in Hindi and Indian Persimmon in English is native to India, however, it is distributed throughout the tropics. AIM OF THE STUDY: As Diospyros malabarica fruit preparation (DFP) possesses medicinal values, the study aims to evaluate its role as natural, non-toxic, and cost-effective dendritic cells (DCs) maturing immunomodulatory agent and also as an epigenetic regulator to combat Non-small cell lung cancer (NSCLC) which is a type of lung cancer whose treatment options such as chemotherapy, radiation therapy, etc. are accompanied with some adverse side effects. Thus, immunotherapeutic strategies are in high demand to evoke tumor protective immunity against NSCLC without causing such side effects. MATERIALS AND METHODS: Peripheral Mononuclear Cells (PBMCs) derived monocytes of normal subjects and NSCLC patients were utilized to generate DCs matured with either LPS (LPSDC) or DFP (DFPDC). Mixed Lymphocyte Reaction (MLR) was carried out with the differentially matured DCs co-culturing T cells and cytotoxicity of lung cancer cells (A549) was measured through LDH release assay and cytokine profiling was carried out via ELISA respectively. PBMCs of normal subjects and NSCLC patients have transfected separately in vitrowith CRISPR-activation plasmid of p53 and CRISPR-Cas9 knockout plasmid of c-Myc to analyze epigenetic mechanism(s) in the presence and absence of DFP. RESULTS: Diospyros malabarica fruit preparation (DFP) treated DC upregulates the secretion of T helper (TH)1 cell specific cytokines (IFN-γ and IL-12) and signal transducer and activator of transcription molecules (STAT1 and STAT4). Furthermore, it also downregulates the secretion of TH2-specific cytokines (IL-4 and IL-10). Diospyros malabarica fruit preparation (DFP) enhances p53 expression by reducing methylation levels at the CpG island of the promoter region. Upon c-Myc knockout, epigenetic markers such as H3K4Me3, p53, H3K14Ac, BRCA1, and WASp were enhanced whereas H3K27Me3, JMJD3, and NOTCH1 were downregulated. CONCLUSION: Diospyros malabarica fruit preparation (DFP) not only increases the expression of type 1 specific cytokines but also augments tumor suppression modulating various epigenetic markers to evoke tumor protective immunity without any toxic activities.

Neem Leaf Glycoprotein in immunoregulation of cancer.

Cancer is a disease having global consequences. Though several new strategies and treatments have been developed so far, they often come with malicious side effects and this paved ways for demand of naturally extracted/driven product as potent anti-cancer agent owing to their reduced toxicity and side effects. One such common Indian household plant Neem (Azadirachta Indica) and its extract have variegated immunomodulatory effects as anti-cancer agent. Neem Leaf Glycoprotein (NLGP) modifies immune cells present in the tumor surroundings as well as in the peripheral system, rather than directly attacking the cancer cells. NLGP acts as a natural immunomodulator showing several functions like sustained tumor growth regulation by stimulating central and effector memory cells as a vaccination adjuvant, normalization of angiogenic activities, controls hypoxia, improves immune evasion techniques as well as suppresses the activity of several immunological cells (Tregs, myeloid-derived suppressor cells, and tumor-associated macrophages) which promote tumor growth and metastasis in the tumor microenvironment (TME). NLGP prioritises type1 immune-microenvironment which consists of T-bet+IFN-γ-producing group 1 innate lymphoid cell (ILC) (ILC1 and natural killer cells), CD8+ cytotoxic T cells (TC1), and CD4+ T helper1 (Th1) cells. In this review we aim to summarize detailed activity of NLGP in cancer immunoregulation.

Neem leaf glycoprotein is nontoxic to physiological functions of Swiss mice and Sprague Dawley rats: histological, biochemical and immunological perspectives.

We have evaluated the toxicity profile of a unique immunomodulator, neem leaf glycoprotein (NLGP) on different physiological systems of Swiss mice and Sprague Dawley rats. NLGP injection, even in higher doses than effective concentration caused no behavioral changes in animals and no death. NLGP injection increased the body weights of mice slightly without any change in organ weights. NLGP showed no adverse effect on the hematological system. Moreover, little hematostimulation was noticed, as evidenced by increased hemoglobin content, leukocyte count and lymphocyte numbers. Histological assessment of different organs revealed no alterations in the organ microstructure of the NLGP treated mice and rats. Histological normalcy of liver and kidney was further confirmed by the assessment of liver enzymes like alkaline phosphatase, SGOT, SGPT and nephrological products like urea and creatinine. NLGP has no apoptotic effect on immune cells but induces proliferation of mononuclear cells collected from mice and rats. Number of CD4(+), CD8(+) T cells, DX5(+) NK cells, CD11b(+) macrophages and CD11c(+) dendritic cells is upregulated by NLGP without a significant change in CD4(+)CD25(+)Foxp3(+) regulatory T cells. Type 1 cytokines, like IFNγ also increased in serum with a decrease in type 2 cytokines. Total IgG content, especially IgG2a increased in NLGP treated mice. These type 1 directed changes help to create an anti-tumor immune environment that results in the restriction of carcinoma growth in mice. Accumulated evidence strongly suggests the non-toxic nature of NLGP. Thus, it can be recommended for human use in anti-cancer therapy.

Neem leaf glycoprotein induces perforin-mediated tumor cell killing by T and NK cells through differential regulation of IFNgamma signaling.

We have demonstrated augmentation of the CD3-CD56+ natural killer (NK) and CD8+CD56_ T-cell-mediated tumor cell cytotoxicity by neem leaf glycoprotein (NLGP). These NK and T cells were isolated from the peripheral blood of head and neck squamous cell carcinoma patients with a state of immunosuppression. NLGP induces TCRalphabeta-associated cytotoxic T lymphocyte (CTL) reaction to kill oral cancer (KB) cells. This CTL reaction is assisted by NLGP-mediated up-regulation of CD28 on T cells and HLA-ABC, CD80/86 on monocytes. CTL-mediated killing of KB cells and NK-cell-mediated killing of K562 (erythroleukemic) cells are associated with activation of these cells by NLGP. This activation is evidenced by increased expression of early activation marker CD69 with altered expression of CD45RO/CD45RA. NLGP is a strong inducer of IFNgamma from both T and NK cells; however, IFNgamma regulates the T-cell-mediated cytotoxicity only without affecting NK-cell-mediated one. Reason of this differential regulation may lie within up-regulated expression of IFNgamma-receptor on T-cell surface, not on NK cells. This NLGP-induced cytotoxicity is dependent on up-regulated perforin/granzyme B expression in killer cells, which is again IFNgamma dependent in T cells and independent in NK cells. Although, FasL expression is increased by NLGP, it may not be truly linked with the cytotoxic functions, as brefeldin A could not block such NLGP-mediated cytotoxicity, like, concanamycin A, a perforin inhibitor. On the basis of these results, we conclude that NLGP might be effective to recover the suppressed cytotoxic functions of NK and T cells from head and neck squamous cell carcinoma patients.