Abstract
Background
Azadirachta indica (neem), belongs to the family of Meliaceae plants, is found in the Indian subcontinent. The neem tree is colloquially referred to as the village pharmacy due to its array of biological properties. Every part of the neem tree like its bark, leaves, sap, fruit, seeds, and twigs find a multitude of uses. It is customary to use them for management of skin diseases and various other infections.
The anticancer properties of neem have been studied in the past and these include its ability to modulate the tumour environment, increase the cytotoxic ability of host monocytes and suppress the proliferation of tumour cells. The present review was conducted with the objective of scrutinizing and assimilating data about the usefulness Azadirachta indica in oral cancer from all the previously done work.
Material and methods
A planned review was conducted of all the studies that investigated the role of Azadirachta indica in oral cancer. Literature search was carried out using PubMed, Scopus and Google scholar databases. In addition to electronic searching, hand searching, cross referencing and various internet engines were also used to collect data. The articles were perused and articles not pertinent to our search were omitted.
Results and conclusion
The anticancer properties of neem were evaluated and the active constituents of neem have been demonstrated to unequivocally have preventive and therapeutic potential against oral cancer. Although, greater exploration of the anticancer properties of neem are required in order to effectively integrate it into the routine management of oral cancer.
Keywords: Azadirachta indica, Neem, Oral cancer, Chemoprevention, Anticancer
1. Introduction
Neem (scientifically referred to as Azadirachta Indica) is a common and popular medicinal tree in the tropical parts of the world. In different parts of the world it is referred to by various names such as neem, nim, margosa, Indian lilac, nimmi, limba, limbo, nimba, imba, mambo, vepa, nimbagaha and many more. Its taxonomic position is as follows1:
Order Rutales
Suborder Rutineae.
Family Meliaceae (mahogany family).
Subfamily Melioideae.
Tribe Melieae
Genus Azadirachta.
Species Azadirachta indica.
The neem tree seeds, bark, fruits, leaves, etc. have been used in various preparation or extracts, most popular being the neem leaf ethanolic extracts and neem liminoids. The biological properties of these neem components have been assessed in different neoplastic and no neoplastic conditions. There is a lack of understanding of the effects of individual components of neem. In this review article, we have analysed all the research work exploring the role of Azadirachta indica in prevention & treatment of oral squamous cell carcinoma. An effort has been made to identify the neem constituents/formulations used for prevention and management of oral cancer and elucidate their mechanism of action.
2. Materials and methods
This literature review was conducted to report and assimilate all previously reported work done to assess the role of Azadirachta indica in oral squamous cell carcinoma. A structured search was carried out using divergent subject-unique digital databases. The search engines were scanned using short-string Boolean-phrases which were marked within brackets. Certain ‘‘operators’’ were marked in capital letters between the search phrases (e.g., < Azadirachta indica AND cancer>).2 Various search terms were incorporated in the searches [e.g., ‘‘oral cancer,’’ ‘‘oral squamous cancer,’’ ‘‘Azadirachta indica,’’ “neem,” ‘‘nimbolide’’ and ‘‘oral carcinogenesis’’]. The searches were conducted with all efforts to ensure no bias and without setting any additional limits relating to author, title, language, subjects or dates. The literature search, done using Pub Med, Pub med central, Scopus and Google scholar databases identified articles published till 2019.
While searching through PubMed, the ‘‘Related Articles’’ link was clicked and all the additional articles were also screened. The references cited in the selected articles were also read and included if they were fitting the inclusion criteria of our search.
At this point in time, all the articles collected from various databases were compiled and tabulated chronologically starting from the latest articles to the oldest ones.
Each of the articles was then carefully read and the extraneous articles were removed on the basis of inclusion and exclusion criteria.
Inclusion criteria: Original research work with internal/external controls assessing the role of neem plant species Azadirachta indica in prevention and treatment of oral squamous cell carcinoma were selected.
Exclusion criteria: Review articles, studies without internal/external controls, those using neem plant species other than Azadirachta indica and studies done on neoplasms/diseases other than oral squamous cell carcinoma were excluded from the review.
To illustrate, rejected articles included ones that testified research on oral cancer using plants species other than Azadirachta indica. The same was done for the use of Azadirachta indica in cancers other than oral squamous cell carcinoma.
3. Results
An aggregate of 13 research articles was counted in for this review. On analysing the various models for research on oral squamous cell carcinoma it was found that animal models were used for 10 research articles3, 4, 5, 6, 7, 8, 9, 10, 11, 12 venous blood from patients with histopathologically confirmed OSCC was used in 2 research articles13,14 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. The research articles were read, content analysed and their role in prevention and treatment of oral cancer was tabulated (Table 1).
Table 1.
Implications of neem in prevention & treatment of Oral cancer.
| S. No | Author/year | Neem component | Technique for preparation | Type of sample | Method/technique | Statistical analysis | Findings |
|---|---|---|---|---|---|---|---|
| 1. | Balasenthi S et al. (1999) | Neam Leaf Extract | Dried leaves were steam boiled, passed through falling film evaporator, spray dried | Male Syrian hamsters-4 groups of 6 hamsters each | Biochemical tests | Students t-test | Decreased susceptibility of oral mucosa to lipid peroxidation indicating that lipid has a suppressing effect on cell proliferation in the target organ. |
| 2. | Subapriya R et al. (2004) | Ethanolic Neem Leaf Extract (ENLE) | Air dried powder was mixed with Ethyl alcohol, filtered, concentrated, dried and suspended in normal saline | Male Syrian hamsters-5 groups of 8 hamsters each | Bone marrow micronucleus test, Histopathological examination, Biochemical assays, Absorbance assays | ANOVA & Least significant difference test | Modulation of lipd peroxidation and antioxidant status in iniatiation phase renedering protective effects against DMBA induced genotoxicity & HBP carcinogensis. |
| 3. | Subapriya R et al. (2005) | Ethanolic Neem Leaf Extract (ENLE) | Air dried powder was mixed with Ethyl alcohol, filtered, concentrated, dried and suspended in normal saline | Male Syrian hamsters-4 groups of 6 hamsters each | Histopathology, SDS-PAGE and Western Blot Analysis, Colorimetric Estimation of Caspase 3 Activity, Statistical Analysis | ANOVA & Least significant difference test | Suppressed development of HBP carcinomas, hyperplasia and dysplasia. Reduced the tumour burden. Increased apoptosis in target organ. |
| 4. | Manikandan P et al. (2008) | Crude ethanolic extract (CEE), ethyl acetate fraction (EAF) and methanolic fraction (MF) | Dried, powdered, mixed with ethanolic extract, filtered and fractionated | Male Syrian hamsters-12 groups of 6 hamsters each | Various absorbance assays, Immunohistochemical assay, Biochemical assays, Western blot analysis | ANOVA & Least significant difference test | Decreased incidence of DMBA-induced HBP carcinomas and reduced preneoplastic lesions. The ethyl acetate fraction was more effective than the methanolic fraction in terms of in vitro antioxidant activity as well as in vivo antiproliferative and antiangiogenic effects. |
| 5. | Bose A et al. (2008) | Neem leaf glycoprotein (NLGP) | Shade dried, pulverized, soaked with phosphate buffered saline (PBS), centrifugation and dialysis | Blood from 33 males& 9 females with Head & neck SCC | Flow cytometric analysis, ELISA, R T-PCR, Cytotoxicity Assay In Vitro, Neutralization of Cytokines, Intracellular Assessment of Cytokines, Perforin, and Granzyme B | Unpaired t-test using INSTAT 3 Software | NLGP increases cytotoxic ability of NK cels and T lymphocytes. |
| 6. | Priyadarshini VR et al. (2009) | Nimbolide and Azadirachtin | Both nimbolide and azadirachtin were purchased from SPIC Science Foundation (Tuticorin, India). | Male Syrian hamsters-8 groups of 10 animals each | In vitro free radical scavenging assays, Histopathologic examination, Immunohistochemical analysis, SDS-PAGE and Western Blot analysis, Reverse Transcriptase (RT) reaction | ANOVA & Least significant difference test | Analysis of Nimbolide, Azadirachtin and Ascorbic acid revealed a concentration-dependent antioxidant activity and reducing potential. The order of antioxidant activity and reducing potential: Nimbolide>Azadirachtin>Ascorbic acid. |
| 7. | Harish Kumar G et al. (2009) | Azadirachtin and Nimbolide | Azadirachtin and nimbolide were purchased from SPIC Science Foundation, Tuticorin, India. | Male Syrian hamsters-8 groups of 10 animals each | Immunohistochemistry, Western blot analysis, Reverse transcriptase (RT) reaction and PCR amplification | ANOVA & Least significant difference test | Azadirachtin and Nimbolide inhibited the development of HBP carcinomas by blocking cell proliferation and inducing apoptosis. |
| 8. | Chakraborty K et al. (2010) | Neem leaf glycoprotein (NLGP) | Shade dried, pulverized, soaked with phosphate buffered saline (PBS), centrifugation and dialysis | 3 oral cancer, 1 colon cancer, 1 T-cell leukaemia & 1 breast cancer cell lines; venous blood | Flow cytometry, ELISA, mRNA isolation and reverse transcription-PCR analysis, Chemotaxis assay, Transmigration assay, Cytotoxicity assay, Immunoblot analysis for p38 MAPK signaling, Statistical analysis | Tukey's test and one-way ANOVA with InStat 3 software. | Increased migratory properties and cytotoxicity of monocytes. |
| 9. | Majid MZ et al. (2016) | Neem Leaf Extract | Decoction, Freeze dried, solution prepared and filtered | Two cancerous oral mucosal cell lines(KB &ORL48) | Immunohistochemistry fluorescent staining method (TUNEL) | Statistical analyses were performed using the SPSS version 11.5 software | Azadirachta indica had an apoptotic effect on the cancer cell lines |
| 10. | Sophia J et al. (2016) | Nimbolide | Nimbolide was obtained from M/s Asthagiri Herbal Research Foundation, Chennai, India. | Male Syrian hamsters-5 groups of 6 animals each | Quantitative real-time RT-PCR expression analysis, Western blotting, Colorimetric assay, Cell cycle analysis and TUNEL assay, Immunohistochemistry, Molecular docking | Nonparametric Mann-Whitney test (StatsDirect, United Kingdom). | Decreased tumor burden & Nimbolide is a potent PI3K/Akt inhibitor. |
| 11. | Koshik J et al. (2017) | Nimbolide | Nimbolide was purchased from Asthagiri Herbal Research Foundation, Chennai. | Male Syrian hamsters-6 groups of 6 animals each; oral cancer cell lines SCC131, SCC4 and the endothelial cell line EAhy926 | RT-PCR, miRNA isolation, Western blotting, Immunohistochemistry, Microvascular density (MVD), Molecular docking, Alamar blue assay, Plasmid construction, Transfection | One-way ANOVA followed by Dunnett's test for in vivo and Tukey posthoc test for the in vitro experiments. | Nimbolide upregulates RECK expression in the HBP model & IN OSCC & endothelial cell lines. |
| 12. | Sophia J et al. (2018) | Nimbolide | Nimbolide was obtained from M/s Asthagiri Herbal Research Foundation, Chennai, India. | SCC131, SCC4 cell cultures, Male Syrian hamsters-5 groups of 6 animals each | Absorbance assay, Fluorescence microscopy, Flow cytometry, Confocal microscopy, Colorimetric assay, qRT-PCR, Western blot analysis, Immunohistochemistry | Mann–Whitney test (StatsDirect, United Kingdom) | Augments apoptosis and induces autophagy in cancer cell lines |
| 13. | Morris J et al. (2019) | Super critical CO2 Neem leaf extract (SCNE) and its bioactive compound,NIM | Organically grown Neem were processed with supercritical CO2 extraction technology | Cal27, HSC3, and SCC4 OSCC cell lines; female athymic nude mice | Cell Migration Assay, Protein Expression, Immunofluorescence, Histological analysis, Immunohistochemistry, Cytokine Assay | One-way ANOVA and Bonferroni's post-hoc test. | Anti-inflammatory and antiproliferative effects of SCNE and NIM in OSCC. SCNE and NIM have profound antitumor activity in vivo. |
4. Discussion
Azadirachta indica is a large perennial tree found commonly in the Indian subcontinent. Various neem preparations have been reported to have anticarcinogenic properties. In this review we have analysed the various research articles available in the literature to assess the role of neem in prevention and treatment of oral cancer. Various studies have used different neem products such as neem leaf and flower extracts, neem seed extracts etc.3,4,8,14,15 The most frequently studied products with respect to OSCC are the ethanolic neem leaf extracts, neem leaf glycoprotein, liminoids like Azadirachtin and Nimbolide. Most studies have been done to assess the chemo preventive properties of neem in Hamster buccal pouch −7, 12-dimethylbenz[a]anthracene (DMBA) induced carcinogenic models. The neem leaf aqueous extract is known to exert its chemo preventive effect in the oral mucosa by regulating the enzymatic breakdown of glutathione.16 Neem components exert their anticancer properties through different mechanisms; a few are explained below.
-
1.
Inhibition of cancer cell proliferation
The hallmark of a cancer cell is its ability to multiply in an uncontrolled and continuous manner. Therefore, the ability to prevent cellular proliferation is a mainstay of anticancer treatment modalities. Earlier studies have shown beneficial effects of using neem extracts in inhibiting cancer cell proliferation as illustrated by the effects of neem oil components on the cell membranes of HeLa cervical cancer cells.17 Neem leaf extracts have been shown to demonstrate significant anti-proliferative properties especially in 7, 12-Dimethyl Benz[a]Anthracene (DMBA)-induced hamster buccal pouch models. The proliferating cell nuclear antigen (PCNA) expression is markedly reduced and there is modulation of cytokeratin expression. These findings clearly point towards potential of neem components to suppress proliferation and promote differentiation, contributing to better prognosis.4,18
-
2.
Effects of neem components on cancer cell death
Neem components have been effectively shown to cause cancer cells death by inducing apoptosis and promoting autophagy. Neem seed and leaf extracts have been evaluated for their increased apoptotic effects in various human cancers such as leukaemia19 prostate20 cervical21 and breast.22 In the oral cavity, studies of DMBA-induced hamster buccal pouch oral carcinogenesis models have shown that neem extracts induce a cascade of molecular events that are corresponding with the findings of increased apoptotic cell death, there is an increase in the expression of Bim, Bax, Apaf-1, caspase 8, caspase 3 and PARP cleavage. A concomitant suppression of Bcl-2 expression is also noted.3,4,6
-
3.
Neem modulates the xenobiotic metabolizing enzymes
Xenobiotic metabolizing enzymes are important chemotherapeutic targets for anticancer therapy. Neem components such as neem leaf extracts and liminoids like Azadirachtin and Nimbolide have dual acting in their mechanism of action against cancer causing cells. They inhibit phase 1 (carcinogen activation) enzymes and simultaneously induce activation of phase 2 (carcinogen detoxification) enzymes. This double edged line of attack makes neem extracts advantageous for control of early phase carcinogenesis.7
-
4.
Other anticancer mechanisms of neem components
Neem components deter tumour invasion and suppress angiogenesis. RECK protein is fundamental for regulating tumour markers involved in invasion and neoangiogenesis. Kowshik J et al. (2017) demonstrated that by nimbolide was an effective agent in targeting Reck through decreased miR-21 and HIF-1α expression leading to down regulation of important mediators of invasion and angiogenesis. Thus suggesting nimbolide could play a vital role in formulating an effective treatment strategy in oral cancer.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
5. Conclusion
The prevention and management of OSCC has been a focus area for researchers across the globe. There are numerous clinical treatment options but they all have many potential side effects which sometimes makes the treatment unviable. The use of phytochemicals in this field has great potential as a supplementary aid in therapeutics or even as a standalone regime in prevention of OSCC. Also, the immunomodulatory effects of various neem components point towards its untapped potential for a future therapeutic vaccine. Larger clinical trials are advocated to unleash the immense potential of neem and its constituents in cancer prevention and management.
Declaration of competing interest
None declared.
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