. 2021 Jul 20;12:699044. doi: 10.3389/fphar.2021.699044

TABLE 6.

Pre-clinical studies in emerging chemopreventive phytochemicals/herbal derivatives against HNC.

Bioactive compound/Herbal derivative	Cell type/Model	Test and dosage	Anti-tumor outcome	Molecular outcome	References
PubChem CID (class) source	Cell type/Model	Test and dosage	Anti-tumor outcome	Molecular outcome	References
Genistein	In vitro study:HSC-3 OSCC)	In vitro invasion assay: 27.3 μg/ml for 24 h	• Invasion↓	• VEGF mRNA↓	Myoung et al. (2003)
CID-5280961 (Phenolic)	In vivo study: Female BALB/c nude mice	In vivo assay: 0.5 mg/kg daily	• Gelatinolytic activity↓	• VEGF mRNA↓	Myoung et al. (2003)
Epigallocatechin-3-gallate	In vitro: OC2 (OSCC)	Invasion and migration assays: >40 μM	• No cytotoxic effect	• MMP-2↓, MMP-9↓, and uPA↓	Ho et al. (2007)
CID-65064 (Phenolic)			• Migration↓
Camellia sinensis			• Migration↓
Berberine	In vitro: SCC-4 (tongue OSCC)	Wound-healing assay: 125 μM for 48 h	• Cell migration and invasiveness ↓	• MMP-2↓, MMP-9↓, u-PA↓, FAK↓, p-p38↓, p-JNK↓, p-ERK↓, IKK↓, NF-κB↓	Ho et al. (2009)
CID-2,353 (Phenolic)
Berberis vulgaris
Gypenosides (Terpene)	In vitro: SAS (tongue OSCC)	Wound-healing assay: 180 μM for 48 h	• Cell migration and invasiveness ↓	• NF-κB↓, COX-2↓, ERK1/2↓, MMP-9↓, MMP-2↓, SOS↓, Ras↓, uPA↓, FAK↓, Akt↓	Lu et al. (2011)
Gynostemma pentaphyllum Makino	In vitro: SAS (tongue OSCC)	Wound-healing assay: 180 μM for 48 h	• Cell migration and invasiveness ↓	• mRNA levels of MMP-2↓, MMP-7↓, MMP-9↓	Lu et al. (2011)
Physalis angulate (crude extract)	In vitro: HSC-3 (OSCC), huvec (human umbilical vein endothelial cells)	Wound-healing assay and Trans well assay: ∼5, 10 μg/ml for 12, 24 h respectively	• Cell migration and invasiveness ↓	• MMP-9 ↓, MMP-2 ↓ and u-PA ↓	Hseu et al. (2011)
			• Cell migration and invasiveness ↓	• TIMP-1 ↑, TIMP-2↑, PAI-1 ↑ and PAI-2 ↑
	In vivo: Fertilized chick embryos	CAM assay: 10–20 μg/ml for 48 h	• In-vivo angiogenesis ↓	• VEGF ↓
Selaginella tamariscina (crude extract)	In vitro study:HSC-3 (tongue OSCC)	Scratch-wound assay: ∼75, 50 μg/ml for 12 and 24 h, respectively	• Cell motility ↓	• MMP-2 ↓, MMP-9 ↓	Yang et al. (2013)
				• TIMP-1 ↑, TIMP-2 ↑
				• MMP-9 promoter activity ↓
			• Cell migration and invasiveness ↓	• Binding of CREB, SP-1 and AP-1 to the MMP-2 promoter ↓
			• Cell migration and invasiveness ↓	• Akt phosphorylation ↓
Selaginella tamariscina (crude extract)	In vitro: HONE-1 (NPSCC)	Scratch-wound assay: >25*μg/mL for 24 h	• Cell motility ↓	• MMP-9 ↓	Hsin et al. (2013)
			• Cell migration and invasiveness ↓	• FAK-src phosphorylation ↓
			• Cell migration and invasiveness ↓	• ERK1/2 phosphorylation ↓
Phenethyl isothiocyanate	In vitro: SAS (tongue OSCC)	Matrigel invasion assay: 0.5, 1, 2 μM for 48 h	• EGF-stimulated invasion↓	• MMP-2↓, MMP-9↓	Chen et al. (2013)
CID-16741 (Alkaloid) Brassica oleracea var. italica			• EGF-stimulated invasion↓	• TIMP-1↑, TIMP-2↑
			• No effect on cell viability	• Activation of EGFR↓
				• PDK1↓, P13K (P85) ↓, AKT↓, NF-κB↓, MMP-1↓, MMP-2↓
				• Phosphorylation of p38↑, JNK↑, ERk↑, MAPK signaling pathway↑
Galium verum (crude extract)	In vitro: FADU (hypopharyngeal SCC), HLaC78 (LSCC), MK (mucosal keratinocytes)	In vitro motility assays: Sub-lethal doses of 33.3 μL/ml	• Cell growth ↓	• MMP-9 ↑, TIMP-1 ↑	Schmidt et al. (2014)
Galium verum (crude extract)		In vitro motility assays: Sub-lethal doses of 33.3 μL/ml	• Cell migration and invasiveness ↓	• MMP-9 ↑, TIMP-1 ↑	Schmidt et al. (2014)
Resveratrol	In vitro study: SCC-9 (tongue OSCC)	Wound-healing assay: >25 μM for 24 h	• No cytotoxicity	• MMP9↓	Lin et al. (2015)
CID-445154 (Phenolic)			• Cell motility↓	• Phosphorylation of ERK and JNK↓
Arachis hypogaea			• Cell migration and invasiveness ↓	• MAPK activation↓
Evodiamine	In vitro: HONE1, CNE1 (NPSCC)	Wound-healing assay: ∼25 μM for 24 h	• Cell migration and invasiveness ↓	• mRNA and protein of MMP-2↓	Peng et al. (2015)
CID-442088 (Alkaloid)				• Translocation of NF-κB (p65)↓
Tetradium spp.				• MMP-2↓
Tetradium spp.				• Phosphorylation ERK1/2↓
Nobiletin	In vitro: HONE-1, NPC-BM (NPSCC)	In vitro wound closure: 40 μM for 24 and 48 h	• Cell migration and invasiveness ↓	• MMP-2↓, TIMP-2↑	Chien et al. (2015)
CID-72344 (Phenolic)	In vivo: Male BALB/c nude mice		• In vivo tumor formation and metastasis↓	• NF-κB and AP-1 signaling pathways↓
Citrus reticulata	In vivo: Male BALB/c nude mice		• In vivo tumor formation and metastasis↓	• Phosphorylation of ERK1/2↓—
Lycopene	In vitro: FaDu (hypopharyngeal SCC), Cal-27 (tongue OSCC)	Colony formation: 25 μM for 24, 48 and 72 h	• Cell proliferation↓, colony formation↓	• Bcl-2↓, Bax↓, caspase-3↓, cleaved caspase-9↓	Ye et al. (2016)
CID-394156 (Terpenes)			• Cell invasion↓	• Phosphorylation of AKT↓, ERK↓
Daucus carota subsp. Sativu s			• Cell invasion↓	• PI3K/AKT, MAPK pathways↓
Toona sinensis (crude extract)	In vivo: Male syrian golden hamsters	*In vivo* treatment: 1 g/kg body weight for 4 weeks	• Incidence of SCC↓, epithelial dysplasia↓	• Proteins of survivin↓, XIAP↓, PCNA↓, iNOS↓, and COX-2 ↓	Wang et al. (2016)
Toona sinensis			• Tumor number↓, tumor volume↓, tumor burden↓, severe dysplastic lesions↓
Toona sinensis			• Apoptosis↑
Triptolide	In vitro: CNEn (NPSCC)	Cell clonogenicity: 4 ng/ml with IR at 0, 2, 4 and 8 Gy	• Cell growth↓, colony number↓	• Bax↑	Zhang et al. (2016)
CID-107985 (Terpene)	In vivo: BalB/C nude mice female	In vivo treatment: 0.075 mg/kg per day	• ionizing radiation↑ induces apoptosis	• Proteins phosph-NF-κb p65↓, Bcl-2↓ and VEGF↓
Tripterygium wilfordii	In vivo: BalB/C nude mice female	In vivo treatment: 0.075 mg/kg per day	• Anti-angiogenesis effects	• Proteins phosph-NF-κb p65↓, Bcl-2↓ and VEGF↓
Raspberries (crude extract)	In-vitro study: SCC-9, SAS (tongue OSCC)	Scratch-wound assay: ∼100 μg/ml for 48 h	• No cell viability effect	• MMP-2 mRNA↓, protein level ↓ and enzyme activity↓	Huang et al. (2017)
Rubus idaeus			• Cell migration and invasiveness ↓	• FAK-src phosphorylation ↓
Rubus idaeus			• Metastasis ↓	• ERK1/2 phosphorylation ↓
Tricetin	In vitro: SCC-9, HSC-3 (tongue OSCC), OECM-1 (OSCC)	Boyden chamber assays: >20 µM in 24 h	• Cell migration and invasiveness ↓	• MMP9 enzyme activity↓, MMP9 mRNA expression↓	Chung et al. (2017b)
CID-5281701 (Phenolic)				• MMP9 enzyme activity↓, MMP9 mRNA expression↓
Eucalyptus globulus				• Regulated MAPK signaling pathway by p-JNK1/2↓ and p-p38↓
Eucalyptus globulus				• p38/jnk-MMP9 axis signaling ↓
Raspberries (crude extract)	In-vitro study: HONE-1, NPC-39 and NPC-BM (NPSCC)	Wound-healing assay: 100 μg/ml for 12 and 24 h	• Tumor cell migration ↓	• MMP-2 mRNA↓, protein level ↓ and enzyme activity↓	Hsin et al. (2017)
Rubus idaeus			• Invasive ability ↓	• ERK1/2 phosphorylation ↓
Rubus idaeus			• Invasive ability ↓	• Inhibits MAPK signaling pathway
Quercentin	In vivo: Male syrian hamsters	In vivo treatment: 50 mg/kg for 14 weeks	• DMBA induced carcinogenesis and apoptosis↓	• NF-Kb p50↓, p65↓	Zhang et al. (2017)
CID-4444051 (Phenolic)			• Tumor incidence↓	• DBMA induced Bcl-2↓, Bax↓
Allium cepa			• Tumor incidence↓	• DBMA induced Bcl-2↓, Bax↓
Leucaena leucocephala (crude extract)	In-vitro study: SCC-9, SAS (tongue OSCC)	Scratch-wound assay: ∼20 μg/ml for 6–48 h	• Cell motility ↓	• MMP-2 ↓	Chung et al. (2017a)
			• Cell migration and invasiveness ↓	• ERK and p38 phosphorylation ↓
			• Anti-metastatic activity	• ERK and p38 phosphorylation ↓
Gallic acid	In vitro: NPC-BM1 (NPSCC)	In Vitro matrix invasion: 25 µM in 24 h	• Invasion↓	• mRNA expression and transcription of MMP-1↓	Pang et al. (2017)
CID-370				• MMP-1 promoter↓, AP-1↓ and ETS-1↓, TIMP-1↓
Hamamelidaceae spp.				• p38 MAPK pathway ↓
Salvianolic acid A	In vitro: SCC-9, SCC-25 (tongue OSCC)	Wound healing migration assay: 50 µM for 24, 48 h	• Cell migration and invasiveness ↓	• MMP-2↓, p-c-Raf↓, p-MEK1/2↓, p-ERK1/2↓ protein	Fang et al. (2018)
CID-5281793 (Phenolic)			• Cell migration and invasiveness ↓
Salvia miltiorrhiza			• Anti-metastatic
Bitter melon (crude extract)	In vivo: C57BL/6 mice	In vivo treatment: 4-NQO- 50 μg/ml; BME- 30% v/v, 600 mg/mouse	• No histological abnormality	• PCNA↓	Sur et al. (2018)
Momordica charantia			• Delayed tumor initiation	• GO categories “Keratin filament”↓,“extracellular region”↓, “GTP binding”↓, “extracellular space”↑, “cytokine activity”↑, “immune response”↑, “positive apoptotic process”↑
Momordica charantia			• Incidence of tongue tumor↓
Eclipta prostrata (crude extract)	In vitro: SCC-9, HSC-3, (tongue OSCC) TW2.6 (OSCC)	Boyden chamber assay: ∼100 μg/ml for 24 h	• Cell migration and invasiveness ↓	• MMP-2↓	Liao et al. (2018)
Eclipta prostrata	In vitro: SCC-9, HSC-3, (tongue OSCC) TW2.6 (OSCC)	Boyden chamber assay: ∼100 μg/ml for 24 h	• Oral cancer metastasis↓	• Phosphorylated ERK1/2↓	Liao et al. (2018)
Black raspberries (crude extract)	In vivo: Male F344 mice	In vivo treatment: 4-NQO- 20 μg/ml	• Oral lesion incidence and multiplicity↓	• Aldoa↓, Hk2↓, Tpi1↓, Pgam2↓, Pfkl↓, Pkm2↓	Knobloch et al. (2019)
Rubus occidentalis	In vivo: Male F344 mice	BRB- 5 and 10% w/w for 6 weeks	• Oral lesion incidence and multiplicity↓	• PKA-AMPK pathway genes↓	Knobloch et al. (2019)
Pinosylvin	In vitro: SAS, SCC-9, HSC-3 (tongue OSCC)	In vitro wound closure: ∼20 μM for 2 h	• Cell migration↓	• Enzymatic activity and protein level of MMP-2↓	Chen et al. (2019)
CID-5280457 (Phenolic)				• Enzymatic activity and protein level of MMP-2↓
Gnetum cleistostachyum				• TIMP-2↑, phosphorylation of ERK1/2↓
Duchesnea indica (crude extract)	In vitro study: SCC-9, SCC-14 (tongue OSCC) and TW2.6 (OSCC)	Wound-healing assay: ∼20, 40 μg/ml for 24, 48 h, respectively	• Cell motility ↓	• MMP-2 ↓	Yang et al. (2019)
			• Cell migration and invasiveness ↓	• ERK1/2 phosphorylation ↓
			• MAPK/ERK signaling pathway ↓	• FAK Y397, src, c-raf, and MEK1/2 phosphorylation ↓

Abbreviations: 4-NQO: 4-Nitroquinoline 1-oxide; Akt: Protein kinase B; AMPK: AMP-activated protein kinase; AP-1: Activator protein 1; Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; COX-2: Cyclooxygenase-2; c-Raf: c- Rapidly Accelerated Fibrosarcoma; CREB: cAMP response element-binding protein; DMBA: 7,12-dimethylbenz(a)anthracene; ERK: Extracellular-signal-regulated kinase; FAK-Src: Focal adhesion kinase-Steroid receptor coactivator; GO: Gene ontology; HK2: hexokinase 2; IKK: Inhibitor of nuclear factor-κB (IκB) kinase; iNOS: Inducible nitric oxide synthase; JNK: c-Jun N-terminal kinase; LC3: Microtubule-associated protein 1A/1B-light chain three; LSCC: Laryngeal squamous cell carcinoma; MAPK: Mitogen-activated protein kinase; MMP: Matrix metallopeptidase; mTOR: mammalian target of rapamycin; NF-κB: Nuclear factor kappa light chain enhancer of activated B cells; NPSCC: Nasopharyngeal squamous cell carcinoma; OSCC: Oral squamous cell carcinoma; PI3K: Phosphatidylinositol 3-kinase; PAI-1: Plasminogen activator inhibitor-1; PCNA: Proliferating cell nuclear antigen; PDK1: 3-Phosphoinositide-dependent kinase 1; PFK1: Phosphofructokinase-1; PGAM2: Phosphoglycerate mutase 2; PKA: Adiponectin activates protein kinase A; PKM2: Pyruvate kinase M2; MEK: Mitogen-activated protein kinase; SCC: Squamous cell carcinoma; SP-1: Specificity protein 1; TIMP-1: Tissue inhibitor of metallopeptidase inhibitor 1; Tpi1: Triosephosphate isomerase; u-PA: urokinase-type plasminogen activator; XIAP: X-linked inhibitor of apoptosis protein.