The Highly Pure Neem Leaf Extract, SCNE, Inhibits Tumorigenesis in Oral Squamous Cell Carcinoma via Disruption of Pro-tumor Inflammatory Cytokines and Cell Signaling

Abstract

Oral squamous cell carcinoma (OSCC) is a deadly disease that comprises 60% of all head and neck squamous cell cancers. The leaves of the Neem tree (Azadirachta indica) have been used in traditional Ayurvedic medicine for centuries to treat numerous oral maladies and are known to have significant anti-inflammatory properties. We hypothesize that a highly pure super critical CO2 Neem leaf extract (SCNE) prevents initiation and progression of OSCC via downregulation of intra-tumor pro-inflammatory pathways, which promote tumorigenesis. Hence, we investigated the anticancer effects of SCNE using in vitro and in vivo platforms. OSCC cell lines (SCC4, Cal27, and HSC3) were treated with SCNE while inflammation, proliferation, and migration were analyzed over time. SCNE treatment significantly inhibited OSCC cell proliferation and migration and reduced MMP activity in vitro, suggesting its potential to inhibit tumor growth and metastasis. The preventive effects of SCNE in ectopic xenograft and 4NQO-1 (4-Nitroquinoline-1-oxide) carcinogen-induced mouse models of OSCC were also evaluated. Indeed, xenografted nude mice showed significant reduction of OSCC tumor volumes. Likewise, SCNE significantly reduced the incidence of tongue dysplasia in the 4NQO-1 OSCC initiation model. In both OSCC animal models, SCNE significantly depressed circulating pro-cancer inflammatory cytokines (host and tumor-secreted) including NFkB, COX2, IL-1, IL-6, TNFα, and IFNγ. In addition, we demonstrate that SCNE downregulates STAT3 and AKT expression and activity in vitro. We also demonstrate that the primary active component, nimbolide (NIM), has significant anticancer activity in established OSCC xenografts. Lastly, we show that SCNE induces an M1 phenotype in tumor associated macrophages (TAMS) in vivo. Taken together, these data strongly support SCNE as means of preventing OSCC via downregulation of pro-cancer inflammatory cascades and NIM as a potential new therapy for existing OSCC.

<p>Evaluation of the Effect of an Aqueous Extract of <em>Azadirachta indica</em> (Neem) Leaves and Twigs on Glycemic Control, Endothelial Dysfunction and Systemic Inflammation in Subjects with Type 2 Diabetes Mellitus – A Randomized, Double-Blind, Placebo-Controlled Clinical Study</p>

“…Though no studies have investigated the effect of neem extract on systemic inflammatory markers in diabetic humans, several studies have reported that neem extract can reduce the levels of pro-inflammatory cytokines (IL-6 and TNF-α) in experimentally-inflammation-induced laboratory animals, which supports our findings. [39][40][41][42][43] It is promising to note that most of the outcome measures in the present study are also significant at 8 weeks as well as 4 weeks of treatment with NEEM, compared to the placebo.…”

A review of the anticancer activity of Azadirachta indica (Neem) in oral cancer

“…On analysing the various models for research on oral squamous cell carcinoma it was found that animal models were used for 10 research articles [3][4][5][6][7][8][9][10][11][12] venous blood from patients with histopathologically confirmed OSCC was used in 2 research articles 13,14 and OSCC cell lines were utilized in 4 research articles. 11,12,14,15 Different parts of the neem plant have been implicated as a cure for many ailments. Neem leaf extracts, neem leaf glycoproteins and liminoids have been the mainstay of research for their anticancer properties in OSCC.…”

“…10 Morris J et al (2019) demonstrated that a highly pure neem leaf extract effectively decreased the pro-cancer inflammatory cytokines and caused disruption of cell signaling and cell migration. 11 Neem leaf glycoprotein has also been shown to improve the migratory capability of monocytes and augment the cytotoxic potential of T lymphocytes. 14 Super critical CO2 neem leaf extract (SCNE) and its bioactive compound, NIM have been shown to decrease the various pro tumour inflammatory mediators and also simultaneously modify cellular signalling in OSCC cell lines.…”

“…The use of SCNE or NIM diminished COX2 expression, NFkBp65; downregulated pSTAT3, pAKT, and pERK1/2 in SCC4 cells. 11…”

Targeting Tumor-Associated Macrophages by Plant Compounds

“…In oral squamous cell carcinoma (OSCC) animal models, highly pure super critical CO 2 leaf extract of Azadirachta indica (Neem) induces an M1 phenotype in TAMs in vivo, and the primary active component, nimbolide (a limonoid tetranortriterpenoid with an α,β-unsaturated ketone system and a δ-lactone ring) has significant anticancer activity in established OSCC xenografts [90]. β-Elemene, a widely known sesquiterpene, regulated the polarization of macrophages from M2 to M1, inhibiting the proliferation, migration and invasion of lung cancer cells and enhancing its radiosensitivity [91].…”