Cardamonin as a potential anticancer agent: Preclinical insights and clinical implications

Table 1 The in vitro anticancer activity of cardamonin

Cell lines	Effects	Molecular mechanism	Ref.
Human hepatocellular carcinoma (HepG2)	(1) Inhibited cellular viability; (2) Arrested cell cycle at G1 phase; (3) Induced apoptosis by accumulating mitochondrial ROS, upregulating caspases expression (caspase 3/7, caspase-8 and caspase-9), upregulating expression of proapoptotic proteins (FADD, Fas, TRIAL, HIF-1, cleaved caspase-3) and downregulating expression of antiapoptotic proteins (HSP60, HSP27 and HSP70, XIAP, catalase, clusterin, and surviving); and (4) Inhibited cellular colonization	Deactivated NF-κB signalling pathway by blocking nuclear translocation of NF-κB and increasing phosphorylation of p53	[61]
	(1) Inhibited cellular viability; and (2) Induced apoptosis by cell shrinkage and chromatin condensation	Blocked TSP50-mediated NF-κB signaling pathway by downregulating protein and mRNA expressions of TSP50	[35]
Human mesenchymal triple-negative breast cancer cells (SUM190; TLR3 activation-enhanced CSC phenotypes)	Inhibited mammosphere formation by blocking TLR3 on the induced CSCs by downregulating protein expression of associated stem-cell genes (OCT4 and c-Myc)	Deactivated concurrently Wnt/β-catenin and NF-κB signalling pathways by concurrent inhibition of nuclear translocations of both β-catenin and NF-κB	[27]
Human mesenchymal triple-negative breast cancer (SUM190 and MDA-MB231), SUM190 (inflammatory breast cancer cell line, invasive ductal carcinoma, ER-PR-HER2-/+) and CAMA-1 cells (adenocarcinoma, ER+/PR-, oncogenic mutations in PTEN and p53, in-frame mutation in E-cadherin gene) and MCF-7 cells (tolerant to 5-FU, doxorubicin, or paclitaxel)	(1) Augmented antimetastatic effects of 5-FU, doxorubicin or paclitaxel; (2) Inhibited mammosphere formation through inhibiting cellular colonization and abolishing self-renewal capacity of CSCs; and (3) Downregulated mRNA expression of CSCs-associated genes (ALDH1, SOX2, c-Myc, OCT4, NANOG) and CSCs-associated histone modifier genes (EZH2, SETDB1, SMYD3)	Deactivated NF-κB and STAT3 signalling pathways by downregulating protein expressions of NF-κB/IKβα and STAT3, respectively	[38]
MDA-MB-231 cells (transfected with pGL3-TSP50 promoter plasmid)	(1) Inhibited cellular viability; (2) Arrested cell cycle at G2/M-phase by upregulating expression of CDK inhibitor (p21) in TSP50-overexpressed cells; and (3) Induced mitochondrial-dependent apoptosis as appeared by cells shrinkage and chromatin condensation with DNA fragmentations, upregulating expression of pro-apoptotic proteins (BAX, activated caspase-9, cleaved caspase-3) and downregulating expression of the anti-apoptotic protein BCL-2	Blocked TSP50-mediated NF-κB signaling pathway through downregulating expression of p65 nuclear with downregulating protein and mRNA expression of TSP50	[35]
Triple negative breast cancer cells (BT-549, ER-negative)	(1) Inhibited cellular viability; (2) Arrested cell cycle at G2/M phase; (3) Induced mitochondrial apoptosis by upregulating expression of cytochrome c, proapoptotic proteins (BAX, cleaved caspase 3 and cleaved PARP) and downregulating expression of the antiapoptotic protein BCL-2; (4) Inhibited cellular colonization; and (5) Retarded invasion by blocking EMT through upregulating protein expression of the epithelia marker E-cadherin and downregulating protein expression of the mesenchymal markers N-cadherin and vimentin	Deactivated Wnt/β-catenin signaling pathway by downregulating protein expression of β-catenin and β-catenin downstream targets (cyclin D1, c-Myc, VEGF, and CDK-4)	[37]
Human TNBC cell line (MDA-MB-231)	(1) Inhibited cellular viability; (2) Arrested cell cycle at G2/M phase; (3) Induced mitochondrial apoptosis through accumulating ROS, downregulating expressions of the anti-apoptotic proteins BCL-2 and upregulating expressions of pro-apoptotic proteins (BAX, cleaved caspase-3 and PARP); and (4) Metabolic reprograming through inhibiting HIF-1α by downregulating protein and mRNA expressions of HIF-1α and HIF-1α target genes (PDHK1 and LDHA)	Deactivated mTOR signalling pathway by downregulating protein expressions of mTOR and S6K	[32]
	(1) Inhibited cellular viability; (2) Arresting cell cycle at G2/M phase; (3) Induced mitochondrial apoptosis through accumulating ROS, upregulating expression of pro-apoptotic proteins (cleaved caspase-3, PARP, and BAX) and downregulating expression of the antiapoptotic protein BCL-2; and (4) Activated FOXO3a by upregulating protein expression of FOXO3a and its target genes (p21, p27, Bim), and upstreaming JNK	Activated JNK/mTOR signalling pathway	[33]
Human ER positive breast cancer cells (MCF-7); resistant to Rapamycin and its analogues, mTOR-inhibitors resistant cells)	Inhibited cellular viability of mTOR resistant cells	Deactivated mTOR signaling pathway by downregulating protein expressions of mTOR, S6K1, and raptor	[36]
Human TNBC cells (MDA-231 and MDA-468)	(1) Inhibited cellular viability; and (2) Desensitized cells by blocking tumor resistant through downregulating expression of PD-L1, and CCL2	Deactivated JAK/STAT axis signaling pathway by downregulating mRNA expression of MUC1, JAK1, and STAT3; deactivated NF-κB signaling pathway by downregulating protein expressions of NF-κB1 (p50), NF-κB2 (p52) and Nrf2	[34]
Human colorectal cancer cells (HCT116, lack p53)	(1) Inhibited cellular viability; and (2) Induced apoptosis (early and late stages) by increasing ROS formation, and upregulating expression of antiapoptotic proteins (BID, BAX, cleaved caspase-8, cleaved caspase 9, and cleaved caspase 3 and cleaved PARP), and downregulating expression of antiapoptotic proteins (cIAP-1, cFLIP, XIAP, BCL-2 and surviving)	Activated CHOP and SP1-dependent TRAIL by upregulating expressions of proteins and mRNA of TRAIL-DR4 and TRAIL-DR5 (death receptors) and upregulating protein expressions of CHOP and SP1 (specificity protein 1)	[58]
Human colon adenocarcinoma cells (SW480)	(1) Inhibited cellular viability; and (2) Arrested cell cycle at G2/M phase	Deactivated Wnt/β-catenin signaling pathway by down-regulating protein expressions of β-catenin and expressions of β-catenin dependent genes (c-Myc, and cyclin D1)	[57]
Human colorectal cancer cells (HCT116)	(1) Inhibited cellular viability; (2) Arrested cell growth at G2/M phase; and (3) Induced autophagic cellular death by increasing level of LC3 proteins (LC3-II) and increasing autophagosomes	Activated JNK signaling pathway by increasing phosphorylation of JNK isomers (JNK1 and JNK2) and upregulating protein and mRNA expressions of p53	[54]
Human colorectal cancer cells (SW620)	(1) Inhibited cellular viability; (2) Arrested cell cycle at S phase; and (3) Induced mitochondrial apoptosis by increasing number of apoptotic cells, and increasing ROS generation	Activated JNK signalling pathway by increasing phosphorylation of JNK and p38	[53]
Human colorectal cancer cells (HCT116) (TSP50 expressing, 5-flourouracil- resistant)	Augmented the antiproliferative effect of 5-FU to inhibit cellular viability, induce apoptosis by increasing activity of caspase-3 and -9, upregulating expression of the proapoptotic protein BAX and downregulating expressions of stem-cell associated proteins (c-Myc and OCT4)	Blocked the activated TSP50-mediated by NF-κB signaling pathway through downregulation of protein expressions of cyclin E, TSP50 and NF-κB	[55]
Human colon cancer cells (HT-29 and SW-460)	Inhibited cellular viability through downregulating mRNA expression of the proliferative factor Ki67	Deactivated STAT3 signaling pathway by downregulating protein expression of STAT3 and its upstream protein JAK2	[60]
Human colorectal cancer cells (HCT116)	Inhibiting cellular viability	Reversed the inflammatory conditions by downregulating mRNA expression of IL-6 and TNF-α	[59]
Human colorectal adenocarcinoma cells (HT-29) and human colorectal carcinoma cells (HCT116)	(1) Inhibited cellular viability; and (2) Hindered cells migration and invasion	Blocked ADRβ2/EMT by downregulating mRNA expression of ADRβ2 and protein expression of matrix metalloproteases (MMP-2 and MMP-9 N-cadherin)	[56]
Lewis lung carcinoma cells (LLC)	(1) Inhibited cellular viability; and (2) Hindered cells invasion and migration by downregulating protein expression of snail and upregulating protein expression of E-cadherin	Deactivated mTOR signaling pathway by inhibiting phosphorylation of mTOR and S6K1	[71]
Non-small-cell lung cancer (H460)	(1) Inhibited cellular viability; (2) Arrested cell cycle at G2/M phase by downregulating protein expression of cyclin D1 and CDK4; (3) Induced apoptosis by upregulating proapoptotic proteins (BAX and cleaved caspase-3) and downregulating expression of the antiapoptotic proteins BCL-2; (4) Inhibited cellular colonization; and (5) Hindered cells migration and invasion	Deactivated PI3K/Akt/mTOR signaling pathway by downregulating protein expression of Akt and mTOR, modulating EMT by upregulating E-cadherin with N-cadherin) and downregulating EMT promotion transcription factor (ZEB1)	[64]
Human gastric cancer 5-FU resistant cells (BGC-823/5-FU)	(1) Restored inhibitory effect of 5-FU on cellular viability, overcoming P-glycoprotein-mediated resistance by downregulating protein expression of P-glycoprotein as well as blocking P-glycoprotein efflux pump through increasing levels of accumulated intracellular Rh-123; (2) Augmented antiproliferative effect of 5-FU through inducing apoptosis (more increase in number of apoptotic cells); and (3) Arrested cell cycle growth at G1 phase	Deactivated Wnt/β-catenin signaling pathway by downregulating protein expression of β-catenin, mRNA expression of stem cell-associated genes (MDR1, CD44, ALDH1, OCT4, c-Myc), Wnt target genes (β-catenin, TCF4 and cyclinD1) and blocking complex formation of β-catenin/TCF4	[73]
Human multiple myeloma cells (U266 and ARH-77)	(1) Inhibited cellular viability; and (2) Induced apoptosis by upregulating expression of proapoptotic proteins (cleaved caspase-3 and PARP) and downregulating expression of antiapoptotic proteins (BCL-2, BCL-xL, survivin, XIAP, cIAP-1 and cIAP-2)	Deactivated NF-κB signaling pathway by blocking phosphorylation of NF-κB p65, downregulating protein expression of Iκβα and IKKβ and products of NF-κB regulating-genes (ICAM-1, COX-2, VEGF)	[67]
Mouse leukemia cells (WEHI-3)	(1) Inhibited cellular viability; (2) Arrested cell cycle at G0/G1 phase; and (3) Induced mitochondria-dependent apoptosis (early and late stage) by increasing accumulation of mitochondrial ROS, upregulating expression of proapoptotic proteins (BAX, cytochrome c, AIF, Endo G, Apaf-1 and cleaved PARP), downregulating expression of antiapoptotic proteins (BCL-2), upregulating mRNA expressions of DAP, TMBIM4 transmembrane, ATG5, and downregulating mRNA expression of DDIT3, DDIT4, BAG6, BCL-2 L13 and BRAT1	Deactivated mitochondria-dependent and ER stress signaling pathways	[66]
Lymphoma cells (SUDHL-4 and OCI-Ly7)	Inhibited cellular viability	Deactivated mTOR signaling pathway by inhibiting phosphorylation of mTORC1 and its downstream substrate S6K1 and downregulating expression of raptor	[65]
Human prostate tumor cell line (PC-3)	(1) Inhibited cellular viability; and (2) Induced apoptosis by increasing DNA fragmentation	Deactivated NF-κB signaling pathway by downregulating mRNA expression of NF-κB1	[50]
Human androgen independent prostate cancer cells (DU145)	(1) Inhibited cellular viability by down regulating expression of complex cell cycle progression proteins (cyclin D1/CDK4, cyclin E/CDK2), and down regulating mRNA expression of cyclin D1; (2) Induced apoptosis by upregulating expression of proapoptotic proteins (cleaved caspase 3 and cleaved PARP), downregulating expression of the antiapoptotic protein BCL-2 and upregulating protein expression of caspase-8 and 9; and (3) Hindered cells invasion and migration	Deactivated STAT3 signaling pathway by decreasing phosphorylation of STAT3, suppressing activity of STAT3 to bind DNA, nuclear pool depletion of STAT3, while downregulating expression of upstream STAT3 kinase (JAK2) and downregulating protein expression of oncogenes products (BCL-xL, BCL-2, XIAP, VEGF, COX-2, and MMP-9)	[49]
Human melanoma cell (A375)	(1) Inhibited cellular viability; (2) Induced apoptosis by upregulating expression of proapoptotic proteins (cleaved caspase-3 and cleaved PARP); and (3) Hindered cells invasion	No signaling pathway was reported	[18]
	(1) Inhibited cellular viability; (2) Induced apoptosis by upregulating pro-apoptotic proteins expression (BAX, cleaved PARP, cleaved caspase 9, cleaved caspase 8) and downregulating expression of the antiapoptotic proteins BCL-2; and (3) Inhibited cells migration and invasion	Deactivated NF-κB signaling pathway by downregulating protein expression of p65 NF-κB	[70]
Human epithelial ovarian cancer cells (SKOV3 and CoCl2-mimicked hypoxic cells)	(1) Inhibited cellular viability; and (2) Anti-angiogenesis and inhibited HIF 1 by downregulating mRNA and protein expressions of VEGF of HIF-1α and HIF-2α, respectively	Deactivated mTOR by downregulating protein expression of mTOR and S6K1	[47]
Human epithelial ovarian cancer cells (SKOV3, cisplatin sensitive)	(1) Augmented the antiproliferative effect of cisplatin through inhibiting cellular viability, arresting cells growth at G2/M phase, and inducing apoptosis by downregulating protein expression of anti-apoptotic proteins (BCL-2, XIAP and survivin); and (2) Augmented antimetastatic effect of cisplatin through inhibiting cellular colonization	Deactivated mTOR signaling pathway by inhibiting phosphorylation of mTOR and its downstream target S6K	[72]
	(1) Inhibited cellular viability; (2) Induced autophagy by increasing autophagosomes and upregulating protein expression of an autophagy marker LC3-II; and (3) Induced apoptosis by upregulating expression of proapoptotic proteins (cleaved PARP and cleaved caspase-3)	Deactivated mTOR signaling pathway by downregulating protein expression of raptor and decreasing phosphorylation level of S6K1	[44]
	(1) Induced autophagy by evidenced by upregulating protein expression of autophagy markers (LC3-II and LAMP1); and (2) Metabolic programming by decreasing lactate secretion and inhibiting activity of hexokinase and lactate dehydrogenase	Deactivated mTOR signaling pathway by deactivating phosphorylation of mTOR and S6K1	[45]
	Inhibited cellular viability	Deactivated mTOR signaling pathway by inhibiting phosphorylation of mTOR and S6K1, while downregulating protein expression of raptor	[47]
Human epithelial ovarian cancer cells (SKOV3)	(1) Inhibited cellular viability; and (2) Induced autophagy by increasing number of autophagosomes and upregulation protein expression of LC3II (an autophagy marker)	Deactivation mTOR signaling pathway by inhibiting phosphorylation of mTOR and S6K1 through upregulating mRNA expression of DAP1 that negatively regulated autophagy	[41]
	(1) Inhibited cellular viability; (2) Arrested cell cycle at G2/M phase by upregulating expressions of cyclin D1, cyclin B1 and p-histone H3 with downregulating expressions of cyclin A, p-cdc2 and Myt1; (3) Induced apoptosis (early and late) by downregulating expression of antiapoptotic proteins (MCL-1, BCL-2) and upregulating expressions of proapoptotic proteins (BAX, and cleaved caspase 3); and (4) Inhibited cellular colonization	Deactivated mTOR signaling pathway by downregulating protein expressions of mTOR, PRAS40, raptor and RagC, in association with deactivating NF-κB signaling pathway by downregulating protein expression of NF-κB, IKKα and IKKβ	[43]
Paclitaxel-resistant ovarian cancer cells (SKOV3-Taxol)	Enhanced the antiproliferative effect of paclitaxel through enhancing inhibiting cellular viability, arresting cell cycle at G2/M phase, and inducing apoptosis by downregulating mRNA expression of MDR1 and protein expression of P-gp	Deactivated NF-κB signaling pathway by downregulating protein expression of p65 NF-κB	[40]
Ovarian cancer cells (SKOV3) with induced TAMs	(1) Inhibited cellular viability by inhibiting M-2 polarization of TAMs through downregulating protein expressions of pro-tumorigenic factors (MMP2 and MMP9), and mRNA expressions of IL-6 and VEGF-α; and (2) Hindered cells migration and invasion	Deactivation mTOR signaling pathway by inhibiting phosphorylation of mTOR and S6K1 and downregulating expression of raptor; deactivated STAT3 signaling pathway by inhibiting phosphorylation of STAT3	[39]
Human ovarian cancer SKOV3 and A2780 cells	(1) Inhibited cellular viability of both cell lines; (2) Inducing oxidative stress through increasing liberation of cellular ROS (little stronger in SKOV3 than that in A2780); and (3) Hindered SKOV3 cells migration through inducing ROS liberation by partial abolishing the reactive oxygen scavenger NAC	Deactivated mTOR signalling pathways pathway by inhibiting phosphorylation of S6K1 at Thr389 and mTORC1 at Ser2448 with downregulating expression of raptor and p-ERK1/2	[48]
Human ovarian SKOV3 and A2780 cells	(1) Inhibited cellular viability; (2) Induced apoptosis by reducing MMP (inducing mitochondrial damage) and downregulating mRNA expression and proteins of SREBP1, FASN, ACC and ACLY; (3) Inhibited activity of CPT-1 (i.e., inhibited lipogenesis); and (4) Inhibited cellular colonization	Deactivated mTOR signalling pathway via raptor by inhibiting phosphorylation of mTOR, S6K1, and 4E-BP1 and expression of raptor	[42]
Pancreas cancer cells (PANC-1 and SW1990)	(1) Augmented the antiproliferative effect of gemcitabine through inhibiting cellular viability, inducing apoptosis by upregulating expression of pro-apoptotic proteins (BAX and cleaved caspase 3), while downregulating expression of the anti-apoptotic protein BCL-2; and (2) Augmented inhibitory effect of gemcitabine on cellular colonization	Deactivated mTOR signaling pathway by inhibiting the phosphorylation of PI3K, AKT, and mTOR; modulated FOXO3a-FOXM1 axis by upregulating expression of FOXO3a and downregulating expression of FOXM1	[63]
Human pancreatic cancer cell line (BxPC3).	(1) Inhibited cellular viability; (2) Induced DNA damaged by enhancing chromatin condensation, nuclear shrinkage and fragmentation though increasing formation of γ-H2AX (one of the histone family member X); (3) Inducing apoptosis by upregulating expression of the pro-apoptotic protein Bax and downregulating anti-apoptotic protein BCL-2, involving iintrinsic or extrinsic pathways as evidenced by increasing expression of cleaved caspase-3 and cytochrome C; and (4) Inhibited cellular colonization	No signaling pathway was reported	[62]
Human esophageal cancer cells (EC-9706 and TE10)	(1) Inhibited cellular viability by downregulating protein expression of the proliferative marker PCNA; (2) Induced apoptosis by increasing dense chromatin-containing cells, proportion of apoptotic cells (at early and late stages), upregulating pro-apoptotic proteins expression (BAD, BAX, cleaved PARP, and cleaved caspase-3) and downregulating expression of anti-the apoptotic protein BCL-2; (3) Inhibited cellular colonization; and (4) Hindered cells invasion and migration through inversing EMT by upregulating protein expression of E-cadherin and downregulating protein expression of N-cadherin, vimentin, snail, MMP2, MMP7, and MMP9	Deactivated PI3K/AKT/mTOR signaling pathway by inhibiting phosphorylation and downregulation of the expression of PI3K and its downstream effector AKT	[30]
Human osteosarcoma cells (143B and MG63)	(1) Inhibited cellular viability; (2) Arrested cell cycle at G2/M phase by downregulating expression of cell cycle-related proteins (cyclin D1, c-Myc, and PCNA); (3) Induced apoptosis by upregulating expression of proapoptotic proteins (BAX, BAD, cleaved caspase 3, and cleaved PARP) and downregulating expression of the antiapoptotic protein BCL-2; and (4) Hindered cells invasion and migration by reversing EMT process through downregulating protein expressions of MMP-2, N-cadherin, snail and vimentin	Activated JNK signaling pathway by increasing phosphorylation of p38 MPAK and JNK	[31]
Human glioblastoma stem cells (CD133+ GSCs)	(1) Inhibited cellular viability by downregulating cell cycle regulator protein (cyclin D1), and VEGF; and (2) Induced apoptosis by upregulating protein expression of intrinsic apoptosis pathway (caspases 3, caspases 9, and PARP), and downregulating expression of antiapoptotic proteins (BCL-2, BCL-xL, MCL-1, surviving)	Deactivated STAT3 signaling pathway by decreasing phosphorylation level of STAT3, and downregulating expressions of STAT3 downstream proteins (BCL-xL, BCL-2, MCL-1, survivin, and VEGF)	[69]
Human bladder cancer cells (T24 and UM-UC-3)	(1) Iinhibited cellular viability; (2) Inducing apoptosis (no evidence); (3) Blocked glycolysis by decreasing glucose uptake, ATP generation and lactate production; and (4) Induced oxidative stress by increasing liberation of cellular ROS through upregulating Nrf2 and NQO1genes	Deactivated PI3K/AKT/mTOR pathway by reducing phosphorylation of PI3K, AKT and mTOR	[51]
Human bladder cancer cells (HT1376 and HTB-9)	(1) Inhibited cellular viability; (2) Arrested cell cycle growth by blocking transition of G0/G1 phase to S phase; and (3) Induced apoptosis by increasing cellular apoptotic rate and upregulating protein expression of ESR1	Deactivated PI3K/AKT/mTOR signaling pathway by downregulation expression of ESR1	[52]

TLR3: Toll-like receptor 3; CSCs: Cancer stem cells; 5-FU: 5-fluorouracil; HER2: Human epidermal growth factor receptor 2; ER: Estrogen receptor; PR: Progesterone receptor; PTEN: Phosphatase and tensin homolog; TSP: Testes-specific protease; TNBC: Triple-negative breast cancer; mTOR: Mammalian target of rapamycin; TAMs: Tumor-associated macrophages; CD: Cluster of differentiation; ROS: Reactive oxygen species; FADD: Fas-associated via death domain; TRIAL: Tumor necrosis factor-related apoptosis-inducing ligand; HIF: Hypoxia-inducible factor; HSP: Heat shock proteins; XIAP: X-linked inhibitor of apoptosis protein; OCT4: Octamer binding transcription factor 4; ALDH1: Aldehyde dehydrogenase 1; SOX2: SRY-box transcription factor 2; EZH2: Enhancer of zeste homolog 2; SETDB1: SET domain bifurcated 1; SMYD3: SET and MYND domain-containing protein 3; CDK: Cyclin-dependent kinase; BAX: B cell lymphoma 2 associated X protein; BCL-2: B cell lymphoma 2; PARP: Poly(ADP-ribose) polymerase; EMT: Epithelial-mesenchymal transition; PDHK1: Pyruvate dehydrogenase kinase 1; LDHA: Lactate dehydrogenase A; S6K: Ribosomal protein S6 kinase; FOXO3a: Forkhead box O3a; JNK: C-Jun N-terminal kinase; PD-L1: Programmed death-ligand 1; CCL2: C-C motif chemokine ligand 2; BID: BH3-interacting domain death agonist; cIAP-1: Cellular inhibitor of apoptosis protein-1; cFLIP: Cellular FLICE-like inhibitory protein; Rh-123: Rhodamine 123; TCF4: Transcription factor 4; BCL-xL: B-cell lymphoma-extra-large; AIF: Apoptosis-inducing factor; Endo G: Endonuclease G; Apaf1: Apoptotic Protease Activating Factor-1; DAP: Death-associated protein; TMBIM4: Transmembrane B cell lymphoma 2 associated X protein inhibitor motif containing 6; ATG5: Autophagy related 5; DDIT: DNA damage inducible transcript; BAG6: B cell lymphoma 2-associated athanogene; BCL2 L13: B cell lymphoma 2 like 13; BRAT1: BRCA1-associated ATM activator-1; LAMP1: Lysosomal associated membrane protein 1; p-cdc2: Phosphorylation-cell division cycle 2; Myt1: Myelin transcription factor 1; MCL-1: Myeloid cell leukemia 1; P-gp: P-glycoprotein; NAC: N-acetyl-L-cysteine; SREBP1: Sterol regulatory element binding protein 1; FASN: Fatty acid synthase; ACC: Acetyl-CoA carboxylase; ACLY: Adenosine triphosphate-citrate lyase; CPT-1: Carnitine palmitoyltransferase 1; PCNA: Proliferating cell nuclear antigen; BAD: B cell lymphoma 2-associated death; p38 MAPK: P38 mitogen-activated protein kinase; ATP: Adenosine triphosphate; NQO1: NAD(P)H quinone dehydrogenase 1; ESR1: Estrogen receptor 1; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; mRNA: Messenger RNA; STAT: Signal transducer and activator of transcription; IKβα: Inhibitor kappa beta alpha protein; VEGF: Vascular endothelial growth factor; JAK: Janus kinase; Nrf2: Nuclear factor erythroid-2-related factor 2; CHOP: C/EBP homologous protein; SP1: Specificity protein 1; TRAIL: Tumor necrosis factor-related apoptosis-inducing ligand; DR: Death receptor; IL: Interleukin; TNF: Tumor necrosis factor; ADRβ2: Adrenoceptor beta 2; MMP: Matrix metalloproteinase; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; ZEB1: Zinc finger E-box binding homeobox 1; IKK: Inhibitor of nuclear factor kappa-B kinase; ICAM-1: Intercellular adhesion molecule-1; COX-2: Cyclooxygenase-2; mTORC1: Mechanistic target of rapamycin complex 1; RagC: Ras-related guanosine triphosphate-binding protein C; 4E-BP1: Eukaryotic initiation factor 4E-binding protein 1; FOXM1: Forkhead box M1.

Table 2 The in vivo animal-based anticancer activity of cardamonin

Cancer type	Model	Cancer induction	Dosage	Mechanism	Ref.
Liver	Athymic nude mice	HepG2-xenograft	Single oral daily of 25 mg/kg or 50 mg/kg for 24 days	Deactivated NF-κB signalling pathway by downregulating protein expression of PCNA, Ki-67 NF-κB-p65 and Ikkβ	[29]
Breast	BALB/c female mic	4T1-pcDNA3-TSP50-luc -xenograft	Single IP dose of 100 μg/50 μL every-three-days for 15 days	Inhibited overexpression of endogenous TSP50	[35]
	Athymic nude mice	SUM190-xenograft	Single IP dose of 25 mg/kg every-other-day for 17 days with doxorubicin	Abolished doxorubicin-enriched CSCs through downregulating mRNA expression of CSCs-associated genes (ALDH1, SOX2, OCT4, NANOG)	[38]
	Athymic nude mice	SUM190 and SUM149-xenograft	Single IP dose of 30 mg/kg every-other-day for 20 days	Blocked TLR3 on the induced-tumorigenic cancer stem cells	[27]
	Female nude mice	MDA-MB-231 cells xenograft	Single daily IP dose of 3 mg/kg for 28 days	Suppressed HIF-1α mediated-cell metabolism	[32]
	Female nude mice	MDA-MB-231 cells xenograft	Single IP dose of 30 mg/kg every-other-day for 28 days	Inducing apoptosis by upregulating expression of p21, p27, and Bim, and increasing the level of cleaved caspase 3, while cyclin D1 level decreased; activated FOXO3a-JNK signalling pathway by upregulating expressions of JNK and FOXO3a	[33]
	BALB/c mic	4T1-xenograft	Single oral daily dose of 5 mg/kg for 30 days	Retarded tumour growth (no further mechanistic information)	[37]
Colorectal	BALB/c nude mice	HT29-xenograft	Single IP dose of 25 mg/kg every-two-days per week for 28 days	Hindered metastasis to lungs by downregulating expression of ADRB2 and matrix metalloproteases (MMP-2 and MMP-9 N-cadherin)	[56]
	C57BL/6 mice	Azoxymethane-induced	Single oral daily dose of 40 mg/kg every-other-day for 90 days	Downregulated expression Ki67; deactivated STAT signaling pathway by downregulating protein expression of JAK2, STAT1, STAT3 and STAT5	[60]
	C57Bl/6 mice	Azoxymethane-induced	Single oral dose of 10 mg/kg five days per week for 30 weeks	Stopped colorectal cancer cells proliferation by downregulating Ki-67 index; deactivated Wnt/β-catenin and NF-κB signaling pathways; modulated expression of microRNAs	[53]
	C57Bl/6 mice	Azoxymethane-induced	Single oral dose of 10 mg/kg either from day 1 to day 140 or from day 50 to day 140	Reduced risk of colitis-induced colon cancer by reducing tumors number, increasing colon shrinkage and alleviating inflammatory insult; blocked accumulation of immune cells; deactivated NF-κB and iNOS signalling pathways; protected mice against inflammation-associated colitis by modulating expression of microRNAs	[59]
Lung (LLC cell)	C57BL/6 mice	LLC-xenograft	Single daily IP dose of 3.5 mg/kg, 7 mg/kg, or 10.5 mg/kg for 20 days	Reduced tumour size; hindered metastasis through inhibiting lung colonization of LLC cells	[71]
Lung (NSCLC)	BALB/c nude mice	H460-xenograft	Single daily IP dose of 5 mg/kg for 14 days	Downregulated expression of Ki-67 in lungs, and deactivated PI3K/Akt/mTOR signalling pathway by downregulating protein expression of Akt and mTOR	[64]
Gastric (5-FU-resistan)	BALB/c nude mice	BGC-823/5-FU xenograft	Single IP dose of 25 mg/kg twice per week for 30 days	Superior retarding tumour growth (no further mechanistic information)	[73]
Esophageal cancer	Male nude mice	EC9706 xenograft	Single oral dose of 5 mg/kg, 15 mg/kg, or 25 mg/kg every-three-days for 26 days	Downregulated protein expression of PCNA, BCL-2, vimentin, PI3K, and Akt; deactivated mTOR signalling pathway	[30]
Pancreas	Athymic BALB/c nude mice	PANC-1 and SW1990 xenograft	Single daily IP dose of 5 μg/kg for 42 days	Modulating FOXO3a/FOXM1 axis pathway through upregulating expression of FOXO3a; deactivated FOXM1/AKT/mTOR signalling pathway by lowering phosphorylation of PI3K, AKT, and mTOR	[63]
Ovarian	Female BALB/c nude mic	SKOV3 or a mix of SKOV3 and M2 macrophages xenograft	Single daily IP dose of 5 mg/kg or 25 mg/kg for 15 days	Downregulated protein expression of Ki67, CD163 and CD206; deactivated mTOR signalling pathway	[39]
	BALB/c nude mice	SKOV3 and PDC xenograft	Single daily IP dose of 20 mg/kg for 13 days	Induced apoptosis; deactivated mTOR and NF-κB signaling pathways	[43]
	Female BALB/c nude mice	SKOV3 xenograft	Single oral daily dose of 15 mg/kg or 30 mg/kg for 20 days	Downregulated expression of Ki-67, raptor and FASN, activated mTOR signaling pathway	[42]
Leukemia	Male BALB/c mice	WEHI-3 xenograft	5 mg/kg, IP, once every two days for 14 days and 42 days	Improved animal survival after 42 days, decreased population of CD3 (T cells), CD11b (monocytes) and macrophages-3 after 14 days, increased population of CD19 (B cells), and enhanced macrophages phagocytic ability	[68]
Lymphoma	Female SCID mice	SUDHL-4 cells expressing mutant; RRAGC xenograft	Single oral daily dose of 15 mg/kg for 30 days	Sensitized RagCT90N-mutant highly to cardamonin	[65]
Osteosarcoma	Female BALB/c nude mice	143B xenograft	Single oral dose of 5 mg/kg, 15 mg/kg, or 25 mg/kg every-two-days for 20 days	Deactivated mTOR signalling pathway, inhibited level of PCNA, BCL-2 and vimentin, and increased phosphorylation level of p38 MAPK and JNK	[31]

NSCLC: Non-small-cell lung cancer; 5-FU: 5-fluorouracil; TSP: Testes-specific protease; IP: Intraperitoneal injection; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; PCNA: Proliferating cell nuclear antigen; IKK: Inhibitor of nuclear factor kappa-B kinase; CSCs: Cancer stem cells; ALDH1: Aldehyde dehydrogenase 1; SOX2: SRY-box transcription factor 2; OCT4: Octamer binding transcription factor 4; TLR3: Toll-like receptor 3; HIF-1: Hypoxia-inducible factor; FOXO3a: Forkhead box O3a; JNK: C-Jun N-terminal kinase; ADRB2: Adrenoreceptor beta 2; MMP: Matrix metalloproteinase; STAT: Signal transducer and activator of transcription; JAK: Janus kinase; iNOS: Inducible nitric oxide synthase; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; mTOR: Mammalian target of rapamycin; BCL-2: B cell lymphoma 2; FOXM1: Forkhead box M1; CD: Cluster of differentiation; FASN: Fatty acid synthase; RagC: Ras-related guanosine triphosphate-binding protein C; p38 MAPK: P38 mitogen-activated protein kinase.

Table 3 Comparative analysis of cardamonin’s in vitro and in vivo anticancer activities across cancer types

Cancer type	Cell studies			Animal studies			Future prospective
	Cells line	IC50 (μM)	Ref.	Induced cancer	Inhibition	Ref.
Liver	HepG2 cells	17.1	[61]	HepG2-xenograft	65.2%	[29]	Promising
		53	[35]
Breast	4T1 Luc cells	85.96	[35]	4T1 Luc-xenograft	Substantial (graphic presentation)	[35]	Not promising in vitro
	MDA-MB-231 cells	45.6	[35]
	MDA-MB-231 cells	25.5	[32]	MDA-MB-231 xenograft	Mild (≤ 50%; graphic presentation)	[32]	Promising
Lung	LCC cells	NR	[71]	LLC-xenograft	84.3%	[71]	Needs in vitro testing
Esophageal	EC9706 cells	8.7	[30]	EC9706-xenograft	Substantial (graphic presentation)	[30]	Promising
Ovary	SKOV3 cells	8.04	[43]	SKOV3 and PDC xenograft	Substantial (graphic presentation)	[43]	Promising
	PDC cells	14.9
	SKOV3 cells	23.4	[42]	SKOV3-xenograft	Substantial (graphic presentation)	[42]	Promising
	A2780 cells	28.8

IC₅₀: Half-maximal inhibitory concentration; NR: Not reported.