Cancer is characterised by uncontrolled cell division and abnormal cell growth, which is largely caused by a variety of gene mutations. There are continuous efforts being made to develop effective cancer treatments as resistance to current anticancer drugs has been on the rise. Natural products represent a promising source in the search for anticancer treatments as they possess unique chemical structures and combinations of compounds that may be effective against cancer with a minimal toxicity profile or few side effects compared to standard anticancer therapy. Extensive research on natural products has shown that bioactive natural compounds target multiple cellular processes and pathways involved in cancer progression.
Cell Lines | Mechanism of Action | Concentration Used | Efficacy/IC50 (Exposure Time) | References | |
---|---|---|---|---|---|
Colorectal cancer | RKO | Inhibit cell proliferation Induce G1 phase cell cycle arrest Induce apoptosis ↓ Bcl-xL; ↑ Caspase-3 & caspase-9 |
0–150 μM | 46.76 μM (68 h) |
[33][25] |
HCT116, HCT116-CH2, HCT116-CH3 | Inhibit cell proliferation Induce G0/G1 & G2/M phase cell cycle arrest: ↓ cyclin D1 & A1; ↑ p53 phosphorylation Induce apoptosis: ↓ Caspase-3; ↓ Bcl-2; ↑ Bax protein |
25 μM Honokiol with 2.5 or 5.0 Gy IR |
N/A | [34][26] | |
HT-29 | Inhibit cell growth & proliferation Induce G1 phase cell cycle arrest: ↓ Cdk1 & cyclin B1 |
0–50 μM followed by 0–5 Gy IR |
23.05 μM (24 h) 13.24 μM (72 h) |
[24][27] | |
HCT116 & SW480 | Inhibit cell proliferation via Inhibition of Notch signalling: ↓ Notch1 & Jagged-1; ↓ Hey-1 & Hes1; ↓ γ-secretase complex; ↓ Skip1 Induce apoptosis: ↑ caspase-3/-7 activity; ↓ Bcl-2 & Bcl-xL; ↑ Bax protein; ↓ cyclin D1 & c-Myc; ↑ p21WAF1 protein Inhibit primary and secondary colonosphere formation |
0–50 μM | N/A | [35][28] |
Cancer Cell Line | Animal Model & Site of Tumour Xenograft | Dose, Duration & Route of Administration | Observation & Mechanism of Action | Efficacy on Tumour Inhibition | References | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Breast cancer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MDA-MB-231 cells | Both flanks of athymic nude mice | 100 mg/kg/day 28 days IP |
Induce tumour growth arrest | Complete arrest of tumour growth from week 2 onwards | [39][32] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MDA-MB-231 cells | Right gluteal region of athymic nude mice | 3 mg/mouse/day Three times a week 28 days IP |
Inhibit tumour progression: ↓ Ki-67; ↑ LKB1 & pAMPK; ↑ ACC phosphorylation, ↓ pS6K & 4EBP1 phosphorylation |
Tumour weight of honokiol-treated group was 0.22 g compared to control group which was 1.58 g | [40][33] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MDA-MB-231-pLKO.1 & MDA-MB-231-LKB1shRNA cells | Right gluteal region of athymic nude mice | 3 mg/mouse/day Three times a week 42 days Oral gavage |
Inhibit cell stemness: ↓ Oct4, Nanog & Sox2; ↓ pSTAT3 & Ki-67 Inhibit mammosphere formation |
Decreased expression of Oct4, Nanog, Sox2 Reduce number of tumour cells showing Ki-67 & pStat3 expression |
[41][34] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RKO & HCT116 | Inhibit cell viability Induce apoptosis: ↑ caspase-3, caspase-8 & caspase-9 activation; ↑ DR5 & cleaved PARP proteins; ↑ survivin protein; ↑ phosphorylated p53 & p53 proteins; ↓ PUMA protein |
0–60 μM | RKO: 38.25 μM (24 h) HCT116: 39.64 μM (24 h) |
[36][29] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Colorectal cancer | Blood cancer | B-CLL | Inhibit cell viability | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RKO cells | Axilla of BALB/c nude mice | Induce apoptosis: ↑ caspase-3 activity; ↑ caspase-8 & caspase-9 activation; ↓ caspase-9; ↑ Bax protein; ↓ Mcl-1 protein | 80 mg/kg/day Treatment on days 8–11, 14–17, 21–24, 28–31 51 days IP0–100 μM |
Inhibit tumour growth Prolong survival of mice49 μM (6 h) 38 μM (24 h) |
[37][30] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
709.9% increase in tumour growth rate in honokiol-treated group compared to 1627.6% and 1408.2% in control and vehicle groups respectively | [ | 33 | ] | [ | 25] | Raji, Molt-4 | Inhibit cell growth: ↓ p65; ↓ NF-κB Induce apoptosis: ↑ JNK activation Increase ROS activity: ↑ Nrf2 & c-Jun protein activation |
0–2.5 μM | Raji: 3.500 μM (24 h) 0.092 μM (72 h) Molt-4: | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
HCT116 cells | 0.521 μM (24 h) | Flank of athymic nude mice | 200 μg/kg/day + 5 Gy irradiation Once a week 21 days IP | [38][31] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Inhibit tumour growth: ↓ CSC proteins → ↓ DCLK1, Sox-9, CD133 & CD44 | Significantly lower tumour weight (<800 mg) in honokiol-IR combination, (~1500 mg) in honokiol treatment group compared to (~3300 mg) in control group | [ | 35 | ] | [28] | Breast cancer | MCF-7, MDA-MB-231, SKBR-3, ZR-75-1, BT-474 | Inhibit cell viability and growth: ↓ EFGR; ↓ MAPK/PI3K pathway activity Induce apoptosis: ↑ PARP protein degradation; ↓ caspase-8; ↑ Bax proteins Induce G1 phase cell cycle arrest: ↓ cyclin D1; ↑ p21 & p27 |
0–100 μM | MCF-7: 40 μM (24 h) MDA-MB-231: 33 μM (24 h) SKBR-3: 29 μM (24 h) ZR-75-1: 39 μM (24 h) BT-474: 50 μM (24 h) |
[39][32] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cervical cancer | MCF-7, MDA-MB-231 | Inhibit cell clonogenicity Inhibit cell anchorage-dependent colony formation Inhibit cell growth, migration & invasion: ↓ pS6K & 4EBP1 phosphorylation; ↑ AMPK activation; ↓ mTORC1 function; ↑ LKB1 & cytosolic localisation |
1–25 μM | N/A | [ | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
KB-8-5 cells | Athymic nu/nu nude mice (site of xenograft not stated) | 50 mg/kg Honokiol Three times a week + 20 mg/kg Paclitaxel Once a week 28 days IP (honokiol) Tail vein injection (paclitaxel) | 40 | ] | Suppress tumour growth: ↓ Ki-67 tissue level [33] |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Induce apoptosis | Significantly lower average tumour volume for honokiol-paclitaxel combination treatment (573.9 mm | 3 | ) compared to control (2585.4 mm | 3) | [31][23] | MCF-7, MDA-MB-231, SUM149, SUM159 | Inhibit cell migration & invasion: ↑ AMPK phosphorylation; ↑ LKB1 Inhibit stem-like characteristics: ↓ Oct4, Nanog & Sox4 protein; ↓ STAT3; ↓ iPSC inducer mRNA |
5 μM | N/A | [41][34] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MCF-7, MDA-MB-231, T47D, SKBR-3, Zr-75, BT-474 | Inhibit cell growth: ↓ PI3K/Akt/mTOR signalling Inhibit cell invasion Induce G0/G1 phase cell cycle arrest: ↓ cyclin D1 & cyclin E; ↓ Cdk2 & c-myc; ↑ PTEN Induce apoptosis: ↑ caspase-3, caspase-6 & caspase-9 activation |
0–40 μM | MCF7: 34.9 μM (24 h) 13.7 μM (48 h) 13.5 μM (72 h) 10.5 μM (96 h) MDA-MB-231: 56.9 μM (24 h) 44.4 μM (48 h) 16.0 μM (72 h) 12.0 μM (96 h) T47D: 47.7 μM (24 h) 41.6 μM (48 h) 17.6 μM (72 h) 7.1 μM (96 h) SKBR-3: 76.1 μM (24 h) 68.1 μM (48 h) 62.7 μM (72 h) 15.7 μM (96 h) ZR-75: 71.1 μM (24 h) 58.1 μM (48 h) 28.7 μM (72 h) 14.5 μM (96 h) BT-474: 80.2 μM (24 h) 65.6 μM (48 h) 39.5 μM (72 h) 15.1 μM (96 h) |
[42][35] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MDA-MB-231 | Inhibit cell proliferation: ↓ c-Src/EGFR-mediated signalling pathway; ↓ c-Myc protein Induce G0/G1 phase cell cycle arrest: ↓ cyclin A, cyclin D1 & cyclin E; ↓ Cdk2, Cdk4 & p-pRbSer780; ↑ p27Kip−1 Induce apoptosis: ↑ caspase-3, caspase-8 & caspase-9 cascade; ↓ Bcl-2 & Bid protein; ↑ PARP cleavage |
0–100 μM | 59.5 μM (72 h) | [43][36] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Lung cancer | A549 | Inhibit cell growth & proliferation Induce G0/G1 phase cell cycle arrest: ↓ Cdk1 & cyclin B1 |
0–50 μM | 12.51 μM (24 h) 7.75 μM (72 h) |
[24][27] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
A549, H460, H226, H1299 | Reduce invasive potential Inhibit PGE2-induced cell migration: ↓ PGE2 production ↓ COX-2 ↑ β-catenin degradation ↓ NF-κB/p65 activity ↓ IKKα |
0–20 μM | N/A | [44][37] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
A549, H1299 | Inhibit cell viability and growth: ↓ class I HDAC proteins; ↓ HDAC activity; ↑ histone acetyltransferase (HAT) activity; ↑ histone H3 & H4 Induce G1 phase cell cycle arrest: ↓ cyclin D1 & cyclin D2; ↓ Cdk2, Cdk4 & Cdk6 |
0–60 μM | N/A | [45][38] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
H460 & A549 | Inhibit cell proliferation Induce apoptosis: ↑ cathepsin D; ↑ cleaved PARP; ↑ caspase-3 Inhibit autophagy: ↑ p62; ↑ LC3-II |
0–60 μM | H460: ~30 μM (48 h) A549: ~40 μM (48 h) |
[46][39] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pc9-BrM3 & H2030-BrM3 (brain metastatic) | Inhibit cell proliferation and cell invasion: ↓ STAT3 protein phosphorylation; ↓ STAT-3 mediated mitochondrial respiratory function | 0–50 μM | PC9-BrM3: 28.4 μM (48 h) H2030-BrM3: 25.7 μM (48 h) |
[47][40] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
H23, A549 & HCC827 | Inhibit cell growth Induce G1 phase cell cycle arrest: ↓EGFR; ↓ class I HDAC; ↓ class IIb HDAC6 activity; ↑ Hsp90 acetylation & EGFR degradation |
0–40 μM | A549: 23.55 μM (24h) |
[48][41] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
H460, A549, H358 | Inhibit cell growth: ↓ c-RAF, ERK & AKT phosphorylation Inhibit colony formation capacity Induce apoptosis: ↑ Bax protein; ↓ Bcl-2 protein; ↑ PARP cleavage Induce G1 phase cell cycle arrest: ↓ cyclin D1; ↑ p21 & p27; ↓ P70S6k kinase activity Induce autophagy: ↑ LC3-I conversion to LC3-II; ↑ Sirt3 mRNA & protein; ↓ Hif-1α protein |
0–80 μM | H460: 30.42 μM (72 h) A549: 50.58 μM (72 h) H358: 59.38 μM (72 h) |
[49][42] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
A549 & 95-D | Inhibit cell viability Induce apoptosis: ↑ ER stress signalling pathway activation; ↑ GRP78, phosphorylation PERK & phosphorylated IRE1α; ↑ cleaved caspase-9 & CHOP; ↓ Bcl-2 protein; ↑ Bax, caspase-3 & caspase-9 Inhibit cell migration |
0–60 μM | N/A | [50][43] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CH27, H460 & H1299 | Inhibit cell growth Induce apoptosis: ↓ Bcl-XL; ↑ mitochondrial cytochrome c release; ↑ BAD protein; ↑ caspase-1, caspase-2, caspase-3, caspase-6, caspase-8 & caspase-9 activity; ↑ PARP cleavage |
0–100 μM | CH27: 40.9 μM (24 h) H460: 41.4 μM (24 h) H1299: 34.7 μM (24 h) |
[25][17] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MSTO-211H | Inhibit cell viability Induce apoptosis: ↑ PARP cleavage; ↑ caspase-3 activation; ↓ Bid & Bcl-xL protein; ↑ Bax protein; ↓ Mcl-1 & survivin protein; ↓ Sp1 Induce G1 phase cell cycle arrest: ↓ cyclin D1 |
0–22.5 μM | N/A | [51][44] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Skin cancer | SK-MEL2 & MeWo | Inhibit cell growth & cell proliferation Induce apoptosis via DNA degradation Induce cell death via mitochondrial depolarization |
0–100 μM | N/A | [52][45] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
A431 | Inhibit cell viability & proliferation Induce G0/G1 phase cell cycle arrest: ↓ cyclin A, cyclin D1, cyclin D2 & cyclin E; ↓ Cdk2, Cdk4 & Cdk6; ↑ p21 & p27 Induce cell apoptosis: ↑ PARP |
0–75 μM | N/A | [53][46] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
B16-F10 | Inhibit cell proliferation Induce cell death: ↑ Autophagosome (vacuoles) formation; ↓ cyclin D1; ↓ AKT/mTOR & Notch signalling |
0–50 μM | N/A | [54][47] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
B16/F-10 & SKMEL-28 | Inhibit cell proliferation & viability: ↓ Notch signalling; ↓ TACE & γ-secretase complex proteins Inhibit clonogenicity Induce G0/G1 phase cell cycle arrest Induce autophagy: ↑ autophagosome formation; ↑ LC3B cleavage Inhibit cell stemness: ↓ CD271, CD166, Jarid1B & ABCB5 |
0–60 μM | N/A | [55][48] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
UACC903 | Inhibit cell growth & proliferation | 0–50 μM | 7.45 μM (24 h) 5.10 μM (72 h) |
[24][27] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SKMEL-2 | Inhibit cell proliferation & viability Induce apoptotic death: ↑ caspase-3, caspase-6, caspase-8 & caspase-9; ↑ PARP cleavage; ↓ procaspase-3, procaspase-8 & procaspase-9 Induce G2/M phase cell cycle arrest: ↓ cyclin B1, cyclin D1, cyclin D2 & PCNA; ↓ Cdk2 & Cdk4; ↑ p21 & p53 |
0–100 μM | N/A | [56][49] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
UACC-62 | Inhibit cell proliferation & viability Induce apoptotic death: ↑ caspase-3, caspase-6, caspase-8 & caspase-9; ↑ cleaved PARP; ↓ procaspase-3, procaspase-8 & procaspase-9 Induce G0/G1 phase cell cycle arrest: ↓ cyclin B1, cyclin D1 & cyclin D2; ↓ Cdk2, Cdk4 & Cdc2p34; ↓ p21 & p27 |
0–100 μM | N/A | [56][49] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Renal cancer | A498 | Inhibit cell proliferation Inhibit colony formation capability Inhibit cell migration and invasion: ↓ Epithelial-mesenchymal transition (EMT); ↓ cancer stem cells (CSC) properties; ↑ miR-141; ↓ ZEB2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Lung cancer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Inhibit cell viability & proliferation: ↓ PI3K/Akt & MAPK/Erk signalling pathways | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Inhibit cell invasion & migration: ↓ MMP2 & MMP9; ↓ NF-κB-mediated E-cadherin pathway Inhibit colony formation Induce apoptosis: ↓ Bcl-2, p-AKT & p-ERK; ↑ Bax protein; ↑ caspase-3 cleavage; ↓ EGFR-STAT3 signalling Reduce spheroid formation: ↓ CD133 & Nestin protein |
0–60 μM | U251: 54.00 μM (24 h) U-87 MG: 62.50 μM (24 h) |
[63][56] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DBTRG-05MG | Inhibit cell growth Induce apoptosis: ↓ Rb protein; ↑ PARP & Bcl-x(S/L) cleavage Induce autophagy: ↑ Beclin-1 & LC3-II |
0–50 μM | ~30 μM | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
H2030-BrM3 cells | Left ventricle of NOD/SCID mice | 2 or 10 mg/kg/day 28 days Oral gavage |
Prevent metastasis of lung cancer cells to brain | 10 mg/kg: Decrease brain metastasis for >70% | [47][40] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
H2030-BrM3 cells | Left lung via left ribcage of athymic nude mice | 2 or 10 mg/kg/day Five days a week 28 days Oral gavage |
Decrease lung tumour growth Inhibit metastasis to lymph node |
10 mg/kg: Significantly reduce incidence of mediastinal adenopathy, decrement of weight of mediastinal lymph node for >80%, only 2/6 mice have lymphatic metastasis | [47][40] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Blood cancer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Raji cells | Back of BALB/c nude mice | 5 mg/20 g & 10 mg/20 g Treatment on days 8–12 & 15–19 20 days (Route of administration not specified) |
Inhibit cell proliferation Inhibit tumour growth |
Tumour growth of honokiol-treated mice was significantly lower (~90 cm3) compared to control mice (~270 cm3) | [38][31] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
HL60 cells | Inoculated intraperitoneally into SCID mice | 100 mg/kg/day Treatment on Day 1–6 47 days IP |
Prolong survival of mice | Median survival time of honokiol-treated mice are longer (37.5 days) compared to vehicle-treated mice (24.5 days) | [85][78] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pancreatic cancer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MiaPaCa cells | Pancreas of immunocompromised mice | 150 mg/kg/day 28 days IP |
Suppress tumour growth Inhibit metastasis: ↓ CXCR & SHH; ↓ NF-κB & downstream pathway Inhibit desmoplastic reaction: ↓ ECM protein; ↓ collagen I |
Significant decrease in tumour growth for honokiol-treated mice (99.6 mm3) compared to vehicle-treated mice (1361.0 mm3) | [58][51] | [64][57] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
U87 MG (Human) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Skin cancer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SKMEL-2 or UACC-62 cells | Right flank of athymic nude mice | 50 mg/kg Three times a week 14–54 days IP |
Decrease tumour growth | SKMEL-2: 40% reduction in tumour volume UACC-62: 50% reduction in tumour volume |
[56][49] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Thyroid cancer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ARO cells | BALB/cAnN.Cg-Foxn1nu/CrlNarl mice (site of xenograft not stated) | 5 or 15 mg/kg/mouse Every three days 21 days Oral gavage |
Decrease tumour volume & tumour weight Induce apoptosis & autophagy |
Control: ~1000 mm3; 700 mg 5 mg/kg Honokiol: ~600 mm3; 400 mg 15 mg/kg Honokiol: ~400 mm3; 200 mg |
[60][53] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nasopharyngeal cancer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
HNE-1 cells | Right dorsal aspect of right foot of BALB/c athymic nude mice | Active-targeting nanoparticles-loaded HK (ATNH), Non-active-targeting nanoparticles-loaded HK (NATNH), Free Honokiol (HK) 3 mg/mouse/day Every three days Euthanise 50% mice after 12 days, rest are left to observe tumour growth & survival time up to 60 days; IV |
Inhibit tumour progression, Induce apoptosis Potential inhibitor of angiogenesis & proliferation |
Efficiency in tumour delay: ATNH > NATNH > Free HK Median survival time: Control: 28.5 days Free HK: 34 days NATNH: 42.5 days ATNH: 57.5 days |
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Bone cancer | Inhibit tumoursphere formation | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
HOS cells | Dorsal area of BALB/c-nu mice | 40 mg/kg/day 7 days IP |
Reduce tumour growth Induce apoptosis & autophagy: ↑ cleaved caspase-3; ↑ LC3B-II & phosphor-ERK (ROS/ERK1/2 signalling pathway) |
Significant decrease in tumour volume & weight of honokiol-treated mice (200 mm3; 0.2 g) compared to control group (~500 mm3; 0.5 g) Increased number of TUNEL-positive cells |
[26][18] | 0–80 μM | ~12 μM (72 h) | [57] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
LM8-LacZ cells | Left flank of C3H/HeNCrl mice | 150 mg/kg/day 25 days; IP | [ | 50 | Inhibit metastasis] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Mean number of micrometastases decreased significantly by 41.4% in honokiol-treated mice compared to control mice | [ | 68 | ] | [ | 61] | Cervix cancer | KB-3-1, KB-8-5, KB-C1, KB-V1 | Inhibit cell viability: ↓ EGFR-STAT3 signalling Induce mitochondria-dependent & death receptor-dependent apoptosis: ↓ Bcl-2, Mcl-1 & survivin; ↑ PARP & caspase-3 cleavage; ↑ mitochondrial release of cytochrome c; ↑ DR5 Enhances in vitro cytotoxicity of Paclitaxel |
0–75 μM | KB-3-1: 12.56 μM (72 h) KB-8-5: 12.08 μM (72 h) KB-C1: 11.40 μM (72 h) KB-V1: 10.39 μM (72 h) |
[ | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Oral cancer | 31 | ] | [ | 23 | ] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pancreatic cancer | MiaPaCa & Colo-357 | Suppress plating efficiency of cells Reduce anchorage-independent clonogenicity growth Suppress migration and invasion ability |
0–5 μM | N/A | [58][51] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
[ | 30 | ] | [ | 22 | ] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SAS cells | Right flank of BALB/cAnN.Cg-Foxn1nu.CrlNarl nude mice | 5 mg/kg or 15 mg/kg Treatment on day 1, 4, 7, 10, 13, 16, 19, 22 35 days Oral |
Reduce tumour growth & volume | Significantly reduction in tumour growth in honokiol-treated mice 29% reduction (5 mg/kg; 21 days), 40% reduction (15 mg/kg; 21 days) 41% reduction (5 mg/kg; 35 days), 56% reduction (15 mg/kg; 35 days) |
[26][18] | MiaPaCa & Panc1 | Inhibit cell growth Induce G1 phase cell cycle arrest: ↓ cyclin D1 & cyclin E; ↓ Cdk2 & Cdk4; ↑ p21 & p27 Induce apoptosis: ↓ Bcl-2 & Bcl-xL proteins; ↑ Bax protein; ↓ IKB-α phosphorylation; ↓ NF-κB constitutive activation |
0–60 μM | MiaPaCa: 43.25 μM (24 h) 31.08 μM (48 h) 18.54 μM (72 h) Panc1: 47.44 μM (24 h) 34.17 μM (48 h) 21.86 μM (72 h) |
[59][52] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Thyroid cancer | ARO, WRO | Inhibit cell growth & proliferation: ↓ ERK, JNK & p37 activation and expression; ↓ mTOR & p70S6K Inhibit colony formation Induce apoptosis: ↑ PARP cleavage; ↑ caspase-3, caspase-8 & PARP activation; ↓ PI3K/AKT & MAPK pathways Induce G0/G1 cell cycle arrest: ↓ cyclin D1; ↓ Cdk2 & Cdk4; ↑ p21 & p27 Induce autophagy & autophagy flux: ↑ LC3-II |
ARO & WRO: 0–60 μM SW579: 0–40 μM |
ARO: | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Prostate cancer | 36.3 μM (24 h) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
C4-2 cells | Bilateral tibia of BALB/c nu/nu athymic nude mice | 100 mg/kg/day 42 days IP |
Inhibit cell proliferation: ↑ Ki-67 Induce apoptosis: ↑ M-31 Inhibit angiogenesis: ↑ CD-31 | 40.1 μM (48 h) 44.8 μM (72 h) WRO: 37.7 μM (24 h) 31.8 μM (48 h) 30.7 μM (72 h) SW579: 19.9 μM (24 h) 10.5 μM (48 h) 8.8 μM (72 h) |
Lower PSA value in honokiol-treated mice compared to control group[60][53] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
[ | 76 | ] | [ | 69 | ] | Nasopharyngeal cancer | HNE-1 | Inhibit cell growth Induce apoptosis Induce G1 phase cell cycle arrest |
0–150 μM (Honokiol & ATNH—Active targeting nanoparticles-loaded honokiol) | Honokiol: | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
PC-3 cells | Left & right flanks above hind limb of nude mice | 1 or 2 mg/mice Monday, Wednesday & Friday two weeks before tumour implantation and duration of experiment after implantation 77 days Oral gavage |
Inhibit tumour growth Inhibit cell proliferation Inhibit neovascularisation | 144.71 μM (24 h) ATNH: 69.04 μM (24 h) |
[30][22] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brain cancer | U251 | Inhibit cell growth Inhibit cell proliferation Induce apoptosis |
0–120 μM | 61.43 μM (24 h) | [61][54] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
T98G | Inhibit cell viability Inhibit cell invasion Induce cell apoptosis: ↑ Bax protein; ↓ Bcl-2; ↑ Bax/Bcl-2 ratio |
0–50 μM | N/A | [62][55] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GBM8401 (Parental) & GBM8401 SP |
Inhibit cell proliferation & viability Induce sub-G1 phase cell cycle arrest Induce apoptosis: ↓ Notch3/Hes1 pathway |
0–20 μM | BMEC (Mouse) |
Inhibit cell viability Inhibit epithelial-mesenchymal transition (EMT): ↓ Snail, β-catenin & N-cadherin; ↑ E-cadherin Inhibit cell adhesion & invasion: ↓ VCAM-1; ↓ phosphor-VE-cadherin-mediated BMEC permeability |
0–20 μM | U87MG: | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brain cancer | 22.66 μM (24 h) | BMEC: 13.09 μM (24 h) |
[65][58] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
U21 cells | Right flank of athymic nude mice | 20 mg/kg Twice a week 27 days Caudal vein injection |
Inhibit tumour growth Inhibit angiogenesis |
U87 MG | Inhibit cell viability Induce G1 phase cell cycle arrest: ↑ p21 & p53; ↓ cyclin D1; ↓ Cdk4 & Cdk6; ↓ p-Rb protein; ↓ E2F1 Induce apoptosis: ↓ procaspase-3; ↑ caspase-8 & caspase-9 activity |
0–100 μM | 52.70 μM | [66][59] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Honokiol-treated mice have significant inhibition of tumour volume by 50.21% compared to vehicle | Significantly lower microvessel present in honokiol-treated cells | [ | 61][54] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
U-87 MG cell suspension pre-treated with honokiol or vehicle for 48h | Yolk sac of Zebrafish larvae | (Concentration N/A) 3 days | Induce apoptosis |
Tumour volume of honokiol-treated mice are significantly lower (~330 mm3; 1 mg), (~50 mm3; 2 mg) compared to control (~400 mm3) | [18][79] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gastric cancer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MKN45 cells | Dorsal side of BALB/c nude mice (nu/nu) | 0.5 mg/kg/day & 1.5 mg/kg/day 10 days Injection (route not stated) |
Inhibit tumour growth: ↓ GRP94 overexpression | 30% reduction in tumour volume (0.5 mg/kg) 60% reduction in tumour volume (1.5 mg/kg) Decreased accumulation of GRP94 |
[86][80] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MKN45 & SCM-1 cells | Peritoneal cavity of BALB/c nude mice | 5 mg/kg Twice a week 28 days IP | GBM8401 (Parental): 5.30 μM (48 h) |
Inhibit metastasis Inhibit angiogenesis |
Honokiol inhibited STAT-3 signalling and VEGF signalling induced by calpain/SHP-1 | [87][81] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Ovarian cancer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SKOV3 cells | Right axilla of BALB/c nude mice | 1 mg liposome-encapsulated honokiol/day 48 days IP |
Inhibit tumour growth Inhibit angiogenesis |
Reduction in tumour growth rate in liposome-encapsulated honokiol-treated mice by 67–70% compared to control | Bone cancer | HOS & U20S | Inhibit cell proliferation Inhibit colony formation Induce G0/G1 phase cell cycle arrest: ↓ cyclin D1 & cyclin E; ↓ Cdk4 Induce mitochondria-mediated apoptosis: ↑ caspase-3 & caspase-9 activation; ↑ PARP cleavage; ↓ Bcl-2, Bcl-xL & survivin; ↑ ERK activation; ↓ proteasome activity; ↑ ER stress and subsequent ROS overgeneration; ↑ GRP78 Induce autophagy: ↑ Atg7 protein activation; ↑ Atg5; ↑ LC3B-II |
0–30 μM | HOS: 17.70 μM (24 h) U20S: 21.50 μM (24 h) |
[67][60] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SAOS-2, HOS, 143B, MG-63 M8, HU09, HU09 M132 Dunn, LM5, LM8 & LM8-LacZ (Mouse) |
Inhibit cell metabolic activity Inhibit cell proliferation Inhibit cell migration Induce rapid cell death via Honokiol-provoked vacuolation |
0–150 μM | (72 h) SAOS-2: 48.38 μM HOS: 51.38 μM 143B: 41.63 μM MG-63M8: 34.88 μM HU09: 59.25 μM HU09M132: 31.88 μM (72 h) Dunn: 36.00 μM LM5: 30.00 μM LM8: 31.13 μM |
[68][61] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Injection of cells into zebrafish | Inhibit cell proliferation | Inhibit cell migration |
Reduced number of cell mass compared to vehicle-treated cells | [63][56] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
U-87 MG cells | Right flank near upper extremity of nude mice | 100 mg/kg/day Treatment at days 1–7 21 days IP |
Reduce tumour growth: ↓ EGFR, pSTAT3, CD133 & Nestin |
Increased number of apoptotic cells in honokiol-treated tissue, Significantly lower tumour volume & tumour weight in honokiol-treated mice | [63][56]
2.2. In Vivo StudiesBased on the in vivo studies, honokiol possessed the capability to inhibit tumour growth, metastasis, and angiogenesis using different animal models, as shown in Table 2. The degree of tumour inhibition was shown to be significantly effective against each distinct cancer cell line, ranging from 0–150 mg/kg via various delivery methods of honokiol between oral gavage or injection (intraperitoneal, caudal vein, or intravenous). Honokiol was shown to downregulate the expression of Oct4, Nanog, and Sox2 which were known to be expressed in osteosarcoma, breast carcinoma and germ cell tumours [41][ 3. Mechanism of Action of Honokiol3.1. Dual Induction of Apoptotic and Necrotic Cell DeathApoptosis is a normal physiological process that maintains the homeostatic cellular balance in multicellular organisms [89][83]. Generally, apoptosis can be classified into two central pathways, namely the intrinsic pathway (mitochondrial-mediated pathway) and extrinsic pathway (death receptor-mediated pathway) [90][84]. The intrinsic pathway is associated with changes in mitochondrial membrane permeability that lead to imbalance in Bax/Bak ratio and release of cytochrome c and other mitochondrial proteins into cytosol [89,90][83][84]. The released cytochrome c interacts with apoptosis protease-activating factor 1 (Apaf1) and forms an apoptosome complex [91][85], which promotes the activation of caspase-9 and later caspase-3, initiating the caspase cascade, which executes cell death in a coordinated way [91][85]. For the extrinsic pathway, the binding of ligands such as tumour necrosis factor (TNF), Fas ligand (Fas-L), and TNF-related apoptosis-inducing ligand (TRAIL) to their respective death receptors (type 1 TNF receptor (TNFR1), Fas (also called CD95/Apo-1) and TRAIL receptors will turn procaspase-8 into active caspase-8 to induce apoptosis [91,92,93][85][86][87]. Honokiol has been shown tp initiate caspase-dependent apoptotic pathways in different types of cancer (Table 1). Chen et al. [14] found that JJ012 human chondrosarcoma cells lose their mitochondrial membrane potential when treated at 10 µM of honokiol, thus leading to apoptosis. Other studies have also shown that honokiol markedly disrupted the balance of Bax/Bcl-2 ratio [13,18,34,63,94,95,96,97][13][79][26][56][88][89][90][91]. The increasing ratio of proapoptotic to antiapoptotic Bcl-2 family proteins (Bax/Bcl-2) will induce the release of cytochrome c and other apoptogenic proteins through the mitochondrial membrane to the cytosol, subsequently leading to the activation of caspase cascade and apoptosis [34][26]. Furthermore, honokiol downregulated the expression of several other anti-apoptosis mRNA and proteins such as Bcl-xL [13[13][79][17][57],18,25,64], survivin [67,98][60][92], and MCL-1 [18][79], as well as upregulated other pro-apoptotic proteins such as BAD, BAX, and BAK proteins [18,25][79][17]. Moreover, honokiol has been shown to effectively induce apoptosis in p53-deficient cancer cells, such as MDA-MD-231 breast cancer cells, as well as lung and bladder cancer cell lines by inhibiting the activation of ras-phospholipase D [39,99,100][32][93][94]. Besides p53, other tumour suppressor genes that will be activated in honokiol treatment include p21 [53][46], p21/waf1 [101][95], p27 [53][46], p38 MAPK [102[96][97],103], and p62 [26,46][18][39]. Besides the intrinsic pathway, honokiol is capable of targeting death receptors TNF-related apoptosis-inducing ligand (TRAIL) receptors and tumour necrosis factor receptors (TNFR) resulting in a sequential activation of caspase-8 and -3, which cleaves target proteins and then leads to apoptosis [104,105,106][98][99][100]. Activation of the death receptor mediated apoptotic pathway is primarily inhibited by cellular-caspase-8/FADD-like IL-1β-converting enzyme (FLICE) inhibitory protein (c-FLIP), which inhibits caspase-8 activation by preventing the recruitment of caspase-8 to the death inducing signalling complex [106][100]. However, honokiol was able to downregulate c-FLIP through the ubiquitin/proteasome-mediated mechanism, resulting in the sensitisation of non-small cell lung cancer cells to TRAIL-mediated apoptosis [107,108][101][102]. Other than intrinsic and extrinsic pathways, honokiol can also induce apoptosis by the endoplasmic reticulum (ER) stress-induced mechanism. A variety of ER stresses result in unfolded protein accumulation responses [109,110][103][104]. For survival, the cells induce ER chaperone proteins to increase protein aggregation, temporarily halt translation, and activate the proteasome machinery to degrade misfolded proteins. However, under severe and prolonged ER stress, an unfolded protein response activates unique pathways that lead to cell death through apoptosis [111][105]. According to a study by Zhu et al. [50][43], honokiol can upregulate the expressions of ER stress-induced apoptotic signalling molecules such as GRP78, phosphorylated PERK, phosphorylated eIF2α, CHOP, Bcl-2, Bax, and cleaved caspase-9 in human lung cancer cells. Chiu et al. [112][106] found that honokiol also led to an increase in ER stress activity in melanoma cell lines B16F10 (mouse), human malignant melanoma, and human metastatic melanoma. Honokiol activated ER stress and down-regulated peroxisome proliferator-activated receptor-γ (PPARγ) activity resulting in PPARγ and CRT degradation through calpain-II activity in human gastric cancer cell lines [86,113,114][80][107][108] and human chondrosarcoma cells [14]. This was due to the ability of honokiol to upregulate and bind effectively to the glucose regulated protein 78 (GRP78) to activate apoptosis [14,115][14][109]. However, this was opposed by another study where treatment of various human gastric cancer cells with honokiol led to the induction of GRP94 cleavage but did not affect GRP78 [86][80]. Necrosis is known as unprogrammed cell death whereby cell swelling and destabilisation of the cell membrane results in the leakage of cellular cytoplasmic contents into the extracellular space, thus causing inflammation [116][110]. Besides apoptosis, honokiol has also been found to induce necrotic cell death in MCF-7 (40 μg/mL honokiol) [117][111], human oesophageal adenocarcinoma cells CP-A and CP-C [118][112], and primary human acute myelogenous leukemia HL60 [85][78] via p16ink4a pathway by targeting cyclophilin D to affect several downstream mechanisms. This phenomenon was also observed in transformed Barrett’s and oesophageal adenocarcinoma cells when treated with honokiol (<40 µM) by targeting their STAT3 signalling pathway, thus resulting in a decrease of Ras activity and phosphorylated ERK1/2 expression [119][113]. The phosphorylation of Ser727 STAT3 induces translocation towards the mitochondria followed by ROS production, ultimately leading to the induction of necrosis [120][114]. Taken together, honokiol demonstrates the dual induction of apoptotic and necrotic cell death. 3.2. Cell Cycle ArrestCancer is attributed to uncontrolled proliferation resulting from abnormal activity of different cell cycle proteins. Therefore, cell cycle regulators are becoming attractive targets in cancer therapy. Honokiol can induce cell cycle arrest in several types of cancer cells, such as in lung squamous cell carcinoma [121][115], prostate cancer cells [75[68][116],122], oral squamous cancer [70][63], UVB-induced skin cancer [123][117], and more as listed in Table 1, by generally inducing G0/G1 and G2/M arrest. This arrest is associated with the suppression of cyclin-B1, CDC2, and cdc25C in honokiol-treated human gastric carcinoma and human neuroglioma cells [97[91][118][119],124,125], downregulation of cyclin dependent kinase (CDK)-2 and CDK-4, and the upregulation of cell cycle suppressors p21 and p27 in human oral squamous cell carcinoma (OSCC) cells [26,97][18][91]. In addition, the downregulation of c-Myc and class I histone deacetylases was also identified as other contributors to cell cycle arrest at the G0/G1 phase in prostate cancer cells [97,122][91][116] and acute myeloid leukemia respectively [44,101,108][37][95][102]. 3.3. AutophagyAutophagy is an evolutionary conserved catabolic process that involves the delivery of dysfunctional cytoplasmic components for lysosomal degradation [126,127][120][121]. The activation of autophagy promotes cell survival and regulates cell growth during harsh and stressful conditions via a reduction of cellular energy requirements by breaking down unnecessary components [82,127][75][121]. In cancer cells, autophagy facilitates both tumour suppression and tumourigenesis by the induction of cell death and tumour growth promotion, respectively [128,129][122][123]. The regulation of mTORC complexes mTORC1 and mTORC2 is involved in controlling the autophagic process. The activation of mTORC1 plays an important role in phosphorylation of autophagy-related protein (ATG) and subsequently inhibiting autophagy, whereas the inhibition of mTORC1 complements the autophagic process [130,131][124][125]. The inhibition of mTORC1 complex will concurrently activate Unc-51-like autophagy-activating kinase (ULK) complex, inducing localisation to the phagophore and followed by class III PI3K activation [132,133][126][127]. Beclin-1 was known to play a role in tumour suppression by recruiting several proteins associated with autophagosome elongation and maturation [134][128]. ATGs regulate the autophagosome elongation. For instance, ATG5-ATG12/ATG16L complexes recruit microtubule-associated protein 1 light chain 3 (LC3), followed by conversion of pro-LC3 to active cytosolic isoform LC3 I by ATG4B [135,136][129][130]. Thereafter, the interaction with ATG3, ATG7, and phosphatidylethanolamine (PE) converts LC3 I to LC3 II. The LC3 II enables the autophagosome to bind to degraded substrates and mature autophagosomes are capable of fusing with lysosomes to selectively remove damaged organelles via autophagy [137][131]. Generally, there are two modes of autophagy known as conventional and alternative autophagy. Conventional autophagy (also known as Atg5/Atg7-dependent pathway) involves the activation of Atg5 and Atg7 which are core regulators of autophagy, and then leads to microtubule-associated protein 1A/1B light chain 3 (LC3) modification and translocation from cytosol to the isolation membrane. This LC3 translocation was considered as a reliable hallmark of autophagy. Contradictorily, alternative autophagy occurs independently without involving Atg5 and Atg7, as well as LC3 modification [128,129,137][122][123][131]. The regulation of autophagy in cancer remains controversial as it plays dual roles in tumour suppression and promotion. Autophagy is believed to contribute to the properties of cancer cells stemness, induction of recurrence, and the development of anticancer drugs. However, the actual mechanism of autophagy in cancer remains unclear. Several studies have highlighted the potential of honokiol to induce cell death via autophagy in human prostate cancer cells [77][70], human glioma cells [138][132], NSCLC cells [30][22], and human thyroid cancer cells [60][53]. The activation of Atg5/Atg7-dependent pathways through the upregulation of LC3B-II, Atg5, and Atg7 levels was observed in honokiol-treated osteosarcoma HOS and U2OS cells and leads to the accumulation of autophagic vacuoles [26][18]. According to a study by Chang et al. [64][57], the expression of two critical autophagic proteins, Beclin-1 and LC3, were found to have increased in the honokiol-treated glioblastoma multiforme cells (DBTRG-05MG cell line). Similarly, the expression of autophagosomal marker LC3-II was also increased in Kirsten rat sarcoma viral oncogene homolog (KRAS) mutated cell lines of non-small cell lung cancer (NSCLC). Other signalling pathways are also found to be involved in honokiol-induced autophagy including the involvement of AMPK-mTOR signalling pathway which leads to autophagocytosis through the coordinated phosphorylation of Ulk1 in Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant lung cancer and melanoma cells [55,60,66,97][48][53][59][91]. Besides this, the ROS/ERK1/2 signalling pathway is also believed to play a certain role in honokiol-induced autophagy though ERK activation and the generation of ROS in treated osteosarcoma cells [67,77,97][60][70][91]. All these recent studies have further supported the potential of honokiol in the induction of autophagy in cancer cells. 3.4. Epithelial-Mesenchymal Transition (EMT)Migratory mesenchymal-like cells are involved in embryonic development, tissue repair, and regeneration, as well as several pathological processes like tissue fibrosis, tumour invasiveness, and metastasis [139,140][133][134]. These migratory mesenchymal cells originate from the conversion of the epithelial cells, and this process is known as epithelial-mesenchymal transition (EMT). This plasticity of cellular phenotypes provides a new insight into possible therapeutic interventions in cancer [140][134]. EMT is characterised by the loss of epithelial markers such as cytokeratins and E-cadherin, followed by an increase in mesenchymal markers such as N-cadherin and vimentin [141][135]. The cellular processes of EMT are composed of several key transcription factors (such as TWIST, SNAI1, SNAI2, ZEB1/2) that act in concert with epigenetic mechanisms and post-translational protein modifications to coordinate cellular alterations [139,142][133][136]. The application of gene expression signatures combining multiple EMT-linked genes has proven useful to evaluate EMT as a contributing factor in tumour development in human cancers. However, the EMT process was shown to be incomplete in tumours, venturing in between multiple translational states and expressing a mixture of both epithelial and mesenchymal genes. This hybrid in partial EMT can be more aggressive than tumour cells with a complete EMT phenotype [141][135]. In addition, EMT contributes to tumour metastatic progression and resistance towards cancer treatment, resulting in poor clinical outcomes [140,141][134][135]. Honokiol has been shown to block and inhibit EMT in many cancer cells such as breast cancer, melanoma, bladder cancer, human non-small cell lung cancer, and gastric cancer (Table 1). Honokiol reduced steroid receptor coactivator-3 (SRC-3), matrix metalloproteinase (MMP)-2, and Twist1, preventing the invasion of urinary bladder cancer cells [108,143][102][137]. In addition, honokiol was also capable of inducing E-cadherin and repressing N-cadherin expression, thus inhibiting the EMT process in J82 bladder cancer cells [108,143][102][137]. In breast cancer cells, honokiol inhibits the recruitment of Stat3 on mesenchymal transcription factor Zeb1 promoter, resulting in decreased Zeb1 expression and nuclear translocation [144][138]. In addition, honokiol increases E-cadherin expression via the Stat3-mediated release of Zeb1 from E-cadherin promoter [144][138]. Collectively, many studies have reported that honokiol effectively inhibits EMT in breast cancer cells, evidence has been found to support a cross-talk between honokiol and Stat3/Zeb1/E-cadherin axis [144][138]. On the other hand, EMT is inhibited by modulating the miR-141/ZEB2 signalling in renal cell carcinoma (A-498) [57][50]. Honokiol inhibited the EMT-driven migration of human NSCLC cells in vitro by targeting c-FLIP through N-cadherin/snail signalling as N-cadherin and snail are downstream targets of c-FLIP [145][139]. Twist1, a basic helix-loop-helix domain-containing transcription factor, promotes tumour metastasis by inducing EMT, and can be upregulated by multiple factors, including SRC-1, STAT3, MSX2, HIF-1α, integrin-linked kinase, and NF-κB. The capability of honokiol in targeting Twist1 can be regarded as a promising therapy for metastatic cancer [108,146][102][140]. Honokiol was found to inhibit breast cancer cell metastasis and eliminate human oral squamous cell carcinoma cell by blocking EMT through the modulation of Snail/Slug protein translation [147,148][141][142]. Honokiol markedly downregulated endogenous Snail, Slug, and vimentin expression and upregulated E-cadherin expression in MDA-MB-231, MCF7, and 4T1 breast cancer cells [148][142]. As primary EMT inducers, Snail and Slug dictate the induction of EMT by targeting E-cadherin and vimentin [144,148][138][142]. Furthermore, when cells were treated with honokiol, Snail and Slug expression levels were decreased from 12 h to 24 h in a time-dependent manner, suggesting that honokiol can reverse the EMT process via the downregulation of Snail and Slug in breast cancer cell lines [148][142]. Besides that, EMT was inhibited in human oral squamous cell carcinoma cell via the disruption of Wnt/β-catenin signalling pathway [147][141]. It was reported that the protein levels of mesenchymal markers such as Slug and Snail were markedly suppressed, while β-catenin and its downstream Cyclin D1 were inhibited [147][141]. It is known that β-catenin could mediate EMT [147[141][143],149], which plays a crucial role in cancer invasion and metastasis. The EMT markers such as Snail and Slug are also the target genes of β-catenin [150][144]. Therefore, the suppression of Snail and Slug in honokiol treated human oral squamous cell carcinoma cells was believed to be due to the inhibition of Wnt/β-catenin signalling pathway [147][141]. Similarly, in U87MG human glioblastoma cell and melanoma cells, Snail, N-cadherin and β-catenin expression levels were decreased, whereas E-cadherin expression was increased after honokiol treatment [65,112][58][106]. 3.5. Suppression of Migration, Invasion and Angiogenesis of Cancer CellsMetastasis is known to be the major cause of death in cancer patients [151][145]. It involves the migration and invasion of tumour cells into neighbouring tissues and distant organs via intravasation into blood or lymphatic system [152,153][146][147]. The formation of invadopodium was stimulated by epidermal growth factor (EGF) and is crucial for the degradation of the extracellular matrix and remodelling membrane proteins, promoting metastasis [151][145]. Therefore, one of the important steps in cancer management is to control tumour cell metastasis, especially for early-stage cancer patients [153][147]. Various studies have reported that honokiol has the capability to suppress tumour metastasis in different types of cancer including breast cancer [40[33][142][148],148,154], non-small cell lung cancer [44,155][37][149] ovarian carcinoma cells [28][20], lung cancer [50][43], U251 human glioma, as well as U-87MG and T98G human glioblastoma cell [63,65,94][56][58][88], oral squamous cell carcinoma (OSCC) [26][18], bladder cancer cell [143][137], pancreatic cancer [58][51], renal cell carcinoma [156[150][151],157], and gastric cancer cells [113][107]. For instance, the percentage of invading urinary bladder cancer (UBC) cells was significantly reduced by 67% and 92% upon 2.4 μg/mL and 4.8 μg/mL of honokiol treatment, respectively [143][137]. Similarly, tumour cell migration was inhibited by 38–66% in A549 cells, by 37–62% in H1299 cells, 12% to 58% in H460 cells and 32% to 69% in H226 cells, in a concentration-dependent manner after treatment with honokiol [44][37]. Furthermore, honokiol also demonstrated an inhibitory effect on the expression of matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9 proteins, which play an essential role in the metastatic process of tumour cells, as well as the regulation of angiogenesis in the maintenance of tumour cell survivability [44,63,143][37][56][137]. MMPs are a group of extracellular matrix degrading enzymes that control various normal cellular processes, such as cell growth, differentiation, apoptosis, and migration [153][147]. However, MMP activity was increased in many tumour cells. The overexpression of MMP-2 and MMP-9 are associated with pro-oncogenic events such as neovascularisation, tumour cell proliferation, and metastasis because it can degrade the extracellular matrix, basement membranes, and adhesion molecules (intercellular adhesion molecule, ICAM, and vascular cell adhesion molecule) and become invasive [58,153,158][51][147][152]. The transition from an epithelial-to-mesenchymal (EMT) phenotype facilitates the breakdown of extracellular matrix followed by the subsequent invasion of the surrounding tissues in order to enter the bloodstream and/or lymph nodes, and travel to distant organ sites. Once cells have reached the distant organ sites, they undergo mesenchymal-to-epithelial transition and begin the establishment of distal metastasis by the surviving cancer cells followed by the outgrowth of secondary tumours [58,159][51][153]. Honokiol has been shown to inhibit the invasion of HT-1080 human fibrosarcoma cells and U937 leukemic cells by inhibiting MMP-9 [160][154]. In addition, honokiol also reduced the protein levels of MMP2 and MMP9 in U251 human glioma and U-87 MG human glioblastoma cell lines in a dose-dependent manner [63][56]. The expression of MMP-2 and MMP-9 were also found to be decreased in both honokiol-treated A549 and H1299 cells (NSCLC cell lines), consistent with the decreased nuclear accumulation of β-catenin as both MMP-2 and MMP-9 are the downstream targets of β-catenin [44,161,162][37][155][156]. In the J82 bladder cancer cell, honokiol repressed the expression of SRC-3, MMP-2, and Twist1 genes which were involved in cancer cell invasion [143][137]. Another proposed mechanism for the inhibitory effects of honokiol on invasion and metastasis is through the liver kinase B1 (LKB1)/adenine monophosphate-activated protein kinase (AMPK) axis. Honokiol treatment increased the expression and cytoplasmic translocation of tumour-suppressor LKB1 in breast cancer cells, which led to the phosphorylation and functional activation of AMPK and resulted in the inhibition of cell invasion and metastasis [40,58][33][51]. The activation of AMPK suppresses mTOR signalling, decreasing the phosphorylation of p70 kDA ribosomal protein S6 kinase 1 (p70S6K1) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4EBP1). This will ultimately inhibit the reorganisation of the actin cytoskeleton in cells, subsequently inhibiting cell migration [40][33]. In human renal carcinoma cell (RCC) 786-0, honokiol significantly upregulated the expression of metastasis suppressor gene (KISS-1), genes encoding TIMP metalloproteinase inhibitor 4 (TIMP4), and KISS-1 receptor (KISS-1R). In addition, honokiol markedly suppressed the expression of genes encoding chemokine (C-X-C motif) ligand 12 (CXCL12), chemokine (C-C motif) ligand 7 (CCL7), interleukin-18 (IL18) and matrix metalloproteinase 7 (MMP7). It was proven that honokiol significantly upregulated KISS1 and KISS1R in the 786-0 cells when treated with honokiol since recent studies showed that the activation of KISS1/KISS1R signalling by kisspeptin treatment decreases the motility and invasive capacity of conventional RCC, and overexpression of KISS1 inhibits the invasion of RCC cells Caki-1 [14,163][14][157]. In short, the activation of KISS1/KISS1R signalling by honokiol suppresses the multistep process of metastasis, including invasion and colony formation, in RCC cells 786-0 [163][157]. Angiogenesis is the formation of new blood vessels for supplying nutrients and oxygen to tissues and cells. In tumourigenesis, angiogenesis is important for the development and progression of malignant tumours [164][158]. The endothelial cells in growing cancer are active due to the release of cell growth and motility promoting proteins, creating a network of blood vessels to overcome its oxygen tension [165][159]. Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) are among the factors that play an important role in tumour angiogenesis [153][147]. In human renal cancer cell lines (786-0 and Caki-1), honokiol induced down-regulation of the expression of VEGF and heme oxygenase-1 (HO-1) via the Ras signalling pathway thus inhibit angiogenesis [166,167][160][161]. In retinal pigment epithelial (RPE) cell lines, honokiol inhibited the binding of hypoxia- inducible-factor (HIF) to hypoxia-response elements present on the VEGF promoter, thereby inhibiting the secretion of VEGF protein [168,169][162][163]. This decrement of VEGF levels resulted in reduced proliferation of human retinal microvascular endothelial cells (hRMVECs) [168][162]. Therefore, honokiol is said to possess both anti-HIF and anti-angiogenic properties. In the overexpression of VEGF-D Lewis lung carcinoma cell-induced tumours in C57BL/6 mice, honokiol was shown to significantly inhibit tumour-associated lymphangiogenesis and metastasis. Furthermore, a remarkable delay in tumour growth and prolonged life span in honokiol-treated mice were also observed [170][164]. In another study, honokiol inhibited VEGF-D-induced survival, proliferation, and microcapillary tube formation in both human umbilical vein endothelial cells (HUVECs) and lymphatic vascular endothelial cells (HLECs). These observations are believed to be due to the inhibition in Akt and MAPK phosphorylation and downregulation of VEGFR-2 expressions in HUVECs as well as VEGFR-3 of HLECs [101,160,171][95][154][165]. Collectively, honokiol has been shown to exert direct and indirect effects on tumour suppression via anti-metastasis, anti-angiogenesis, and anti-lymphangiogenesis by mainly affecting HIF- and VEGF/VEGFR- dependent pathways. However, an in-depth mechanism of honokiol on the inhibition of metastatic progression and spread should be further explored in the future. |