Table 1.
In vitro chemopreventive properties of NOB, 5-DMN and NOB-metabolites.
| Compounds | Activities | Cell lines | Treatment/Assay (Treatment Duration) |
Assays/Results/Mechanisms | References |
|---|---|---|---|---|---|
| NOB | Anti-proliferative | HT-29 | H-thymidine uptake assay | - IC50 of NOB = 4.7 μM | [70] |
| - IC90 of NOB = 13.9 μM | |||||
| 5-DMN | - IC50 of 5-DMN = 8.5 μM | ||||
| - IC90 of 5-DMN = 171 μM | |||||
| NOB | Cytotoxicity | COLO320, SW480 and Caco-2 | MTS viability assay (48 h) | - IC50 for COLO320 = 40.4 ± 9.1 μM | [79] |
| - IC50 for SW480 = 245 ± 9.1 μΜ | |||||
| - IC50 for Caco-2 = 305.6 ± 41.9 μΜ | |||||
| Apoptosis-inducing | Apoptosis assays—DNA fragmentation | - DNA ladder pattern | |||
| 200 μΜ—2-fold increase DNA fragmentation in COLO320 | |||||
| - gel electrophoresis (48 h) | |||||
| Anti-proliferative | BrdU labelling index | - 34.7 ± 4.7% BrdU-binding cells at 100 μΜ | |||
| - 44.4 ± 6.4% BrdU-binding cells at 40 μΜ | |||||
| NOB | Anti-metastasis | HT-29 | ELISA | [77] | |
| - proMMP-7 expression | - At 100 μM, no detection of proMMP-7 in media, ~280 pg/mL proMMP-7 in media | ||||
| qPCR and Western blot | - >25 μM, reduced RNA and protein (both intracellular and supernatant) expression of proMMP-7 | ||||
| AP-1 binding activity | - Inhibited binding activity of AP-1 (transcription factor for MMP-7 gene) | ||||
| NOB | Anti-proliferative | HT-29 | Cell counting assay | - IC50 of NOB ≈ 50 μM | [14] |
| - Inhibited cell proliferation in a time- and dose-dependent manner | |||||
| Cell cycle arrest | |||||
| Cell cycle analysis | - Induced G1 phase cell cycle arrest (60 and 200 μM) | ||||
| - Propidium iodide staining | |||||
| Apoptosis-inducing | Apoptosis assay | - No significant apoptosis detected at 60 and 100 μM | |||
| Resumption of proliferation | - Resumed proliferation within 24 h of removal of NOB and achieve the same stage of growth as compared to control after four days of removal of NOB | ||||
| NOB 5-DMN |
Cytotoxicity | HCT116, HT-29 | MTT viability assay (48 h) | - IC50 of NOB on HCT116 = 37 μM | [63] |
| - IC50 of 5-DMN on HCT116 = 8.7 μM | |||||
| - IC50 of NOB on HT-29 = 46.2 μM | |||||
| - IC50 of 5-DMN on HT-29 = 22 μM | |||||
| Cell cycle arrest | Cell cycle analysis - Propidium iodide staining (24 h) Western blot |
- At 8 μM, 5-DMN induced G2/M phase arrest in HCT116 | |||
| - At 36 μM, 5-DMN induced G2/M phase arrest in HT-29 | |||||
| - At 16 μM, NOB reduced CDK-2 expression | |||||
| - At 4 μM and 8 μM, 5-DMN increased p21 and Rb, while decreased CDK-2 and p-Rb. | |||||
| Apoptosis-inducing | Apoptosis assay | - At 8 μM, 5-DMN increased early apoptosis by 2.2-fold in HCT116 | |||
| Annexin-V/PI (48 h) | - At 36 μM, 5-DMN increased early apoptosis by ~2-fold in HT-29 | ||||
| Western blot | - At 16 μM, NOB did not increase apoptotic cell population in HCT116/HT-29 | ||||
| - At 4 μM and 8 μM, 5-DMN increased expressions of cleaved caspase 8, cleaved caspase 3 and cleaved PARP. | |||||
| 5-DMN | Apoptosis-inducing | HCT116 (p53 +/+) and HCT116 (p53 −/−); HCT116 (Bax +/−) and HCT116 (Bax −/−); HCT116 (p21 −/−) |
Apoptosis assay Annexin-V/PI |
- At 15 μM, 5-DMN increased late apoptotic/necrotic cell in HCT116 (p53 −/−) > HCT115 (p53 +/+), suggesting the apoptotic inducing action is independent of p53 | [80] |
| - At 15 μM, 5-DMN increased early apoptotic cell in HCT116 (Bax +/−), but not in HCT116 (Bax −/−) | |||||
| Cell cycle arrest | Cell cycle analysis - Propidium iodide staining |
- At 15 μM, 5-DMN arrested cells at G2/M and G0/G1 phases in HCT116 (p53 +/+) cells, but only caused G2/M phase arrest in HCT116 (p53 −/−) cells | |||
| - G0/G1 is p53 dependent and G2/M is p53-independent | |||||
| NOB; 3′-DMN; 4′-DMN; 3′,4′-DMN |
Cytotoxicity | HCT116, HT-29 | MTT viability assay | - At 2.03 μM and 3.28 μM, NOB and 3′-DMN, respectively showed no significant cytotoxicity against HCT116 and HT-29 | [54] |
| - At 24.13 μM, 4′-DMN inhibited growth of HCT-116 by 45% and HT-29 by 33% | |||||
| - At 12.03 μM, 3’,4’-DMN inhibited growth of HCT116 by 30% and HT-29 by 9% | |||||
| - combination of all three NOB-metabolites inhibited growth of HCT116 by 64% and HT-29 by 62% (no significant difference to three NOB-metabolites + NOB) | |||||
| Cell cycle arrest | Cell cycle analysis - Propidium iodide staining (24 h) |
- NOB (40 μM) arrested cells at G0/G1 phase in both HCT-116 and HT-29 | |||
| - 3′-DMN (40 μM) arrested cells at both S phase and G2/M phase in HCT-116; while arrested cells at both G0/G1 and G2/M phase in HT-29 | |||||
| - 4′-DMN (40 μM) induced a stronger effect than NOB in arresting cells at G0/G1 phase in HCT-116 and HT-29 | |||||
| - 3′,4′-DMN (20 μM) arrested cells at both S phase and G2/M phase in HCT-116; while arrested cells at both G0/G1 and G2/M phase in HT-29 | |||||
| Apoptosis inducing | Western blot | - NOB and all three NOB-metabolites cause profound increase in expression of p21Cip1/Waf1 | |||
| Annexin-V/PI (48 h) | - NOB (40 μM) increased early apoptotic cell population by 3.3-fold, increased late apoptotic cell population by 4.2-fold in HCT116 | ||||
| - 3′-DMN (40 μM) increased early apoptotic cell population by 5.0-fold, increased late apoptotic cell population by 3.5-fold in HCT116 | |||||
| - 4′-DMN (40 μM) increased early apoptotic cell population by 4.9-fold, increased late apoptotic cell population by 7.1-fold in HCT116 | |||||
| - 3′,4′-DMN (20 μM) increased early apoptotic cell population by 7.6-fold, increase late apoptotic cell population by 4.5-fold in HCT116 | |||||
| -3′-DMN (40 μM) and 4’-DMN (40 μM) did not cause significant apoptosis in HT-29 | |||||
| - 3′,4′-DMN (20 μM) exhibits stronger apoptosis effect than NOB (40 μM) in HT-29 | |||||
| Western blot | - NOB (40 μM) only increased activation of caspase-9 and did not affect caspase-3 or PARP levels in HCT116 | ||||
| - NOB-metabolites increased activation of caspase-3, caspase-9 and other downstream proteins like PARP in HCT116 | |||||
| NOB-Met (2.03 μM NOB: 3.28 μM 3′-DMN: 24.13 μM 4′-DMN: 12.03 μM 3′,4′-DMN |
Anti-inflammatory | RAW264.7 | Western Blot | - At 0.5× concentration of NOB-Met, supressed LPS-induced iNOS expression by 56.4% | [76] |
| - At 1× and 2× concentration of NOB-Met, completely abrogated LPS-induced iNOS expression | |||||
| - At ×0.5, increased expression of NQO1 by 21% as compared to LPS-treated cells | |||||
| - At ×1, increased expression of HO-1 by 10%, increased expression of NQO1 by 34% as compared to LPS-treated cells | |||||
| - At ×2, increased expression of HO-1 by 37%, increased expression of NQO1 by 50% as compared to LPS-treated cells | |||||
| - Induced translocation of Nrf2 | |||||
| Cell cycle arrest | HCT116 | Cell cycle analysis - Propidium iodide staining Western blot |
- At 1×, induced G0/G1 phase arrest; while at 2×, induced G0/G1 and G2/M phases arrest | ||
| - Reduced expressions of CDK-2, CDK-4, CDK-6 and cyclin D, while increased expressions of p53 and p27 | |||||
| NOB, 5-DMN | Cytotoxicity | HCT116, HT-29, COLO205 | MTT viability assay | - At 40 μM, NOB significantly reduced viability of HCT116, HT-29 and COLO205 by ~20–30% | [49] |
| - At >5 μM, 5-DMN significantly reduced viability of HCT116, HT-29 and COLO205 | |||||
| Apoptosis inducing | Cell cycle analysis - SubG1 quantification Western |
- At 20 μM, 5-DMN increased apoptosis ratio by ~26%, while no increased in subG1 population in NOB-treated COLO205 | |||
| - At 10 and 20 μM, significantly increased expression of cleaved PARP in COLO205 | |||||
| NOB | Anti-inflammatory | Human synovial fibroblast, mouse macrophage J774A.1 | ELISA | - At >4 μM, NOB inhibited PGE2 induced by IL-1α in human synovial fibroblast | [81] |
| Western blot and qPCR | - At >16 μM, NOB reduced mRNA of COX-2 induced by IL-1α in human synovial fibroblast | ||||
| - At 64 μM, NOB inhibited COX-2 protein expression induced by IL-1α in human synovial fibroblast | |||||
| qPCR | - At 32 μM, NOB reduced mRNA of IL-1α, IL-1β, IL-6, TNF-α induced by LPS in J774A.1 | ||||
| Western blot | - At >16 μM, NOB reduced proMMP-1 and proMMP-3 induced by IL-1α in human synovial fibroblast | ||||
| - At >16 μM, NOB enhanced TIMP-1 expression in response to IL-1α in human synovial fibroblast | |||||
| NOB | Anti-inflammatory | Mouse adipocyte 3T3-L1 | ELISA | - At 50 and 100 μM, NOB suppressed MCP-1 secretion induced by TNF-α IN 3T3-L1 adipocytes | [82] |
| Western blot | - At 50 and 100 μM, NOB reduced ERK phosphorylation in 3T3-L1 adipocytes treated with TNF-α | ||||
IC50—half maximal inhibitory concentration; AP-1—activator protein-1; PI—propidium iodide; PARP—poly (ADP-ribosome) polymerase; CDK—cyclin-dependent kinase; Rb—retinoblastoma; LPS—lipopolysaccharide; iNOS—inducible NO synthase; NQO1—NAD(P)H quinone oxidoreductase 1; HO-1—heme oxygenase-1; PGE2—prostaglandin E2; IL—interleukin; TNF—tumor necrosis factor; TIMP-1—tissue inhibitor metalloprotease-1; MCP-1—monocyte chemoattractant protein-1.