AMPK; Apoptosis

In a dose-dependent manner, treatment with 6-gingerol significantly lowers the LoVo human colon cancer cell's ability to proliferate. In LoVo cells, 6-gingerol strongly induces G2/M phase arrest and only slightly affects sub-G1 phase. Cyclin A, cyclin B1, and CDK1 levels are decreased; however, the negative cell cycle regulators p27Kip1 and p21Cip1 are elevated as a result of 6-gingerol treatment. Furthermore, treatment with 6-gingerol increases p53 phosphorylation and intracellular reactive oxygen species (ROS). As well as the later stages of carcinogenesis, such as angiogenesis and metastasis, 6-gingerol is effective at suppressing the transformation, hyperproliferation, and inflammatory processes that cause and promote carcinogenesis[1]. 6-gingerol has direct cytotoxic effects on cultures of tumor cells, such as colorectal cancer cells, HL-60 cells and breast cancer cells[2].

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6-gingerol inhibits tumor growth in several types of murine tumors, such as B16F1 melanomas, Renca renal cell carcinomas and CT26 colon carcinomas, established by inoculating tumor cells on the flanks of mice, although it does not lead to complete eradication of the tumors. 6-gingerol treatment of tumor-bearing mice results in massive infiltration of CD4 and CD8 T-cells and B220+ B-cells, but decreases the number of CD4+Foxp3+ regulatory T-cells. In 6-gingerol-treated mice, the CD8 tumor-infiltrating T lymphocytes exhibit high levels of IFN-, a CTL CD107a cell activation marker, and chemokine receptors CXCR3 and CCR5 that are expressed on TH1 cells[2].

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Oral administration of 6-Gingerol (3 μg/ml in drinking water, equivalent to ~500 μg/kg/day) to mice bearing CT26 colon carcinomas, Renca renal cell carcinomas, or B16F1 melanomas significantly inhibited tumor growth. Tumor volumes were measured every 2‑3 days; treated mice showed reduced tumor size and about 10 days extended survival, but all mice eventually died from tumors by days 30‑50 [2].
- In mice inoculated with 1×10⁵ CT26 cells, 6-Gingerol treatment reduced tumor incidence: 80% of control mice developed tumors by day 12 versus only 50% in treated mice [2].
- 6-Gingerol increased the percentages and absolute numbers of CD3⁺CD4⁺, CD3⁺CD8⁺ and B220⁺ (B‑cell) tumor‑infiltrating lymphocytes (TILs), while decreasing CD4⁺Foxp3⁺ regulatory T cells, CD11b⁺Ly6G⁺, CD11b⁺F4/80⁺, CD11c⁺ and CD11b⁺Gr1^int innate immune cells in tumors. The total number of TILs per mg tumor tissue was 10‑20‑fold higher in treated mice for CD4, CD8 and B cells, whereas suppressor cell numbers were unchanged. The ratios of effector to suppressor cells (CD4/Foxp3, CD8/Foxp3, CD4/Gr1^int, CD8/Gr1^int) were increased 13‑21‑fold [2].
- 6-Gingerol enhanced IFN‑γ production by CD8 and CD4 TILs. The number of IFN‑γ⁺ CD8 T cells per mg tumor was significantly elevated, and whole tumor tissue IFN‑γ level was 8‑fold higher in treated mice, while TGF‑β level was decreased [2].
- 6-Gingerol increased MHC class I (H‑2K^d) expression on CT26 tumors as shown by immunohistochemistry and FACS. In vitro, IFN‑γ (1 ng/ml) directly up‑regulated H‑2K^d on CT26 cells [2].
- 6-Gingerol treatment induced functional CTL activity: >50% of CD8 TILs from treated mice expressed CD107a upon contact with tumor cells, compared to 11% in controls. The number of CD8⁺CD107a⁺ cells per mg tumor was 3,820±400 in treated vs 91±10 in controls [2].
- 6-Gingerol promoted tumor cell apoptosis as shown by TUNEL staining and Annexin V staining [2].
- 6-Gingerol increased expression of chemokine receptors CXCR5, CCR5 and CXCR3 on CD4 and CD8 T cells in TILs and tumor‑draining lymph nodes (TDLNs) [2].
- In adoptive transfer experiments using OT‑1 CD8 T cells (from mice fed 6-Gingerol or vehicle) into EG7 tumor‑bearing mice, 6-Gingerol‑pretreated OT‑1 CD8 T cells massively infiltrated into tumors and divided, whereas control OT‑1 cells did not. Direct ex vivo preincubation of OT‑1 CD8 T cells with 6-Gingerol followed by adoptive transfer into RAG2^‑/‑ mice also resulted in strong tumor infiltration and proliferation [2].

A cell line for colon cancer The DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% L-glutamine, and 100 μg/mL penicillin/streptomycin is used to maintain LoVo at 37°C in a humid environment with 5% CO2. Approximately 80% confluence is reached after cell growth in 10 cm Petri dishes that were initially seeded with 2 105 cells per mL. After that, the cells are gathered for the subsequent analyses, such as the analyses of cell viability, flow cytometry, and immunoblotting. In order to treat cells with 6-gingerol, cells are first starved for 24 hours (h) in serum-free DMEM, and then they are incubated for 24 or 48 hours with 6-gingerol at a series of concentrations (1, 5, 10, and 15 μg/mL) in the same medium.

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Mice: For 14 days, different oral dosages of [6]-gingerol were administered to mice with colitis brought on by the DSS. The level of proinflammatory cytokines in colon tissues is measured, and body weight and colon inflammation are assessed[2].

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Female Balb/c and C57BL/6 mice (7 weeks old) were subcutaneously inoculated with tumor cells (CT26: 3.0×10⁵ cells; Renca: 7×10⁵ cells; B16F1: 2.5×10⁵ cells; EG7: 3×10⁶ cells) on the left flank. Three days after tumor inoculation, mice were given drinking water containing 6-Gingerol (0.3, 3.0 or 30 μg/ml) or vehicle (0.01% ethanol). Fresh drug‑containing water was provided every 2‑3 days. Based on average water consumption (3‑4 ml/day per 20 g mouse), the doses were approximately 50, 500 or 5000 μg/kg/day. The 3.0 μg/ml regimen (500 μg/kg/day) was used in all subsequent experiments. Tumor diameters were measured every 2‑3 days, and tumor volume calculated as (major axis)×(minor axis)×(height)×0.52. Animals were euthanized when tumors exceeded 20 mm in longest dimension [2].
- For adoptive transfer experiments, donor OT‑1 mice were fed 6-Gingerol (3 μg/ml) or vehicle for 2 days. CD8 T cells were purified from lymph nodes, labeled with 2 μM CFSE, and 3×10⁶ cells were injected intravenously into recipient mice bearing 10‑day‑old EG7 tumors. Tumors and TDLNs were collected 1, 2, and 3 days after transfer. In another experiment, naive OT‑1 CD8 T cells were preincubated with 6-Gingerol ex vivo before adoptive transfer into EG7 tumor‑bearing RAG2^‑/‑ mice [2].

[1]. 6-Gingerol Inhibits Growth of Colon Cancer Cell LoVo via Induction of G2/M Arrest

[2]. Int J Cancer . 2012 Jun 1;130(11):2618-28.

Gingerol is a β-hydroxy ketone, a product of 5-hydroxydecone with 4-hydroxy-3-methoxyphenyl substitution at the 1-position; it is believed to have an inhibitory effect on lipogenesis. It is one of the components of fresh ginger. Gingerol has antitumor activity and is also a plant metabolite. It is a β-hydroxy ketone belonging to the guaiacol class of compounds. It has been reported that gingerol is found in cumin (Cuminum cyminum), African ginger (Aframomum melegueta), and several other organisms with relevant data. See also: ginger (partial); (S)-6-gingerol (note moved here).

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6-Gingerol (1‑[4′‑hydroxy‑3′‑methoxyphenyl]‑5‑hydroxy‑3‑decanone) is the major pungent principle of ginger (Zingiber officinale). It has been reported to have cancer‑preventive activities, direct cytotoxic effects on some tumor cell lines (colorectal cancer, HL‑60, breast cancer), and to inhibit pulmonary metastasis of B16F1 melanomas and skin papillomas in mice. However, no previous study had examined its effect on pre‑established tumors. This paper is the first to show that 6-Gingerol enhances antitumor immune responses by increasing TIL infiltration [2].
- The final goal of cancer immunotherapy is to achieve antitumor responses that eradicate established tumors. 6-Gingerol may be useful in adoptive cell therapy to promote infiltration of tumor‑reactive TILs. The effective dose in mice (500 μg/kg/day) extrapolates to human consumption of only 10‑30 g of ginger per day. Ginger is widely used as a spice with low toxicity [2].
- The paper suggests that the increased B‑cell infiltration in 6-Gingerol‑treated mice might suppress complete tumor eradication; ongoing studies are testing B‑cell depletion combined with 6-Gingerol [2].

C17H26O4

294.3859

294.183

C, 69.36; H, 8.90; O, 21.74

23513-14-6

23513-14-6

442793

White to off-white solid

1.1±0.1 g/cm3

453.0±35.0 °C at 760 mmHg

30 - 32ºC

159.0±19.4 °C

0.0±1.2 mmHg at 25°C

1.523

2.48

2

4

10

21

293

1

O([H])[C@]([H])(C([H])([H])C(C([H])([H])C([H])([H])C1C([H])=C([H])C(=C(C=1[H])OC([H])([H])[H])O[H])=O)C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H]

NLDDIKRKFXEWBK-AWEZNQCLSA-N

InChI=1S/C17H26O4/c1-3-4-5-6-14(18)12-15(19)9-7-13-8-10-16(20)17(11-13)21-2/h8,10-11,14,18,20H,3-7,9,12H2,1-2H3/t14-/m0/s1

(5S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one

6-Gingerol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50~58 mg/mL (169.8~197 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.49 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.49 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.49 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (84.92 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)