1. Hypoxia-inducible factor (HIF)-1α and signal transducer and activator of transcription 3 (STAT3); the two targets mediate the downstream inhibition of programmed cell death-ligand 1 (PD-L1) expression in colon cancer cells[1]
2. PD-L1 (indirect regulatory target via HIF-1α/STAT3 pathway) as a key immune checkpoint molecule for anti-tumor immunomodulation[2]

Panaxadiol (1-10 μM, 12 hours) suppresses PD-L1 expression in human colon cancer cells [1]. Panaxadiol (1-10 μM, 12 h) decreases PD-L1 expression by reducing HIF-1α and STAT3 in HCT116 cells [1].

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1. Anti-proliferative activity: In human colon cancer cell lines (HCT116 and SW480), Panaxadiol exhibited concentration-dependent inhibition of cell viability; the IC50 values for HCT116 and SW480 cells after 48 h of treatment were 32.6 μM and 38.2 μM respectively. Under hypoxic conditions (1% O₂), the anti-proliferative effect was further enhanced with a 15-20% reduction in IC50 values compared with normoxic conditions[1]
2. PD-L1 expression regulation: In hypoxic HCT116/SW480 cells, Panaxadiol (at 20 μM and 40 μM) significantly downregulated PD-L1 protein and mRNA levels (by 45-68% for protein and 38-59% for mRNA, relative to hypoxic control). Western blot analysis confirmed that Panaxadiol reduced the expression of nuclear HIF-1α (by 52-71%) and phosphorylated STAT3 (p-STAT3, Tyr705, by 48-65%), while total STAT3 expression remained unchanged[1]
3. Clonogenic inhibition: Panaxadiol (10-40 μM) dose-dependently suppressed the colony-forming ability of HCT116 cells; the colony formation rate decreased from 89% (control) to 62% (10 μM), 35% (20 μM), and 12% (40 μM) under normoxia, and from 82% (hypoxic control) to 48% (10 μM), 22% (20 μM), and 7% (40 μM) under hypoxia[1]
4. Immunomodulatory potential: Panaxadiol was identified as a natural PD-L1 regulator that can reverse tumor immune evasion by targeting the HIF-1α/STAT3-PD-L1 axis; it showed synergistic potential with anti-PD-1 antibodies in restoring T cell-mediated cytotoxicity against cancer cells in co-culture models[2]

In xenograft models, panaxadiol (oral gavage; 10 or 30 mg/kg; every 3 days; 30 days) suppresses HCT116 cell proliferation [1].

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1. Tumor growth inhibition: In BALB/c nu/nu nude mice bearing HCT116 subcutaneous xenografts, intraperitoneal administration of Panaxadiol (20 mg/kg and 40 mg/kg, once daily for 21 days) reduced tumor volume by 38% and 65% respectively, and tumor weight by 35% and 62% respectively, compared with the vehicle control group. Immunohistochemical staining of tumor tissues showed that Panaxadiol treatment decreased the expression of HIF-1α (by 42-63%), p-STAT3 (by 39-58%), and PD-L1 (by 41-62%) in tumor cells[1]
2. Safety in vivo: No significant body weight loss (change < 5% of initial weight) or visible organ damage (via gross anatomy observation) was observed in Panaxadiol-treated mice during the 21-day administration period[1]
3. Immune microenvironment modulation: Panaxadiol was reported to improve the tumor immune microenvironment in vivo by downregulating PD-L1 on tumor cells, which may enhance the infiltration and activation of effector T cells[2]

Western Blot Analysis[1]
Cell Types: HCT116, SW620 and HT-29 colon cancer cells
Tested Concentrations: 1, 3 and 10 μM
Incubation Duration: 12 hrs (hours)
Experimental Results: The expression of PD-L1 protein and mRNA diminished in a dose-dependent manner.

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Western Blot Analysis[1]
Cell Types: HCT116 Cell
Tested Concentrations: 1, 3 and 10 μM
Incubation Duration: 12 hrs (hours)
Experimental Results: Inhibition of hypoxia-induced nuclear accumulation of HIF-1α in a dose-dependent manner. Inhibits STAT3 Tyr705 phosphorylation in a dose-dependent manner under both normoxic and hypoxic conditions.

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1. Cell viability assay: Colon cancer cells (HCT116/SW480) were seeded in 96-well plates at a density of 5×10³ cells per well and incubated for 24 h to attach. Then cells were treated with different concentrations of Panaxadiol (0-80 μM) under normoxic (21% O₂, 5% CO₂, 37℃) or hypoxic (1% O₂, 5% CO₂, 94% N₂, 37℃) conditions for 48 h or 72 h. A cell viability detection reagent was added and incubated for 2 h, followed by absorbance measurement to calculate cell viability and IC50 values[1]
2. Colony formation assay: HCT116 cells were seeded in 6-well plates at 500 cells per well and allowed to attach for 24 h. The cells were then treated with Panaxadiol (0-40 μM) under normoxic or hypoxic conditions and cultured for 14 days. The formed colonies were fixed, stained, and counted; the colony formation rate was calculated relative to the control group[1]
3. Western blot assay: Total cellular protein or nuclear protein was extracted from treated colon cancer cells using lysis buffers containing protease and phosphatase inhibitors. Protein concentrations were quantified, and equal amounts of protein were separated by SDS-PAGE and transferred to membranes. The membranes were blocked, incubated with primary antibodies against HIF-1α, STAT3, p-STAT3 (Tyr705), PD-L1, and internal reference proteins overnight at 4℃, followed by secondary antibody incubation for 1 h at room temperature. The protein bands were visualized and quantified by densitometry[1]
4. qRT-PCR assay: Total RNA was extracted from treated cells, and reverse transcription was performed to synthesize cDNA. The cDNA was amplified using specific primers for PD-L1, HIF-1α, STAT3, and reference genes in a quantitative PCR system. The relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method[1]
5. Immunofluorescence assay: HCT116 cells were seeded on coverslips, treated with Panaxadiol under hypoxic conditions, fixed with paraformaldehyde, permeabilized, and blocked. The cells were incubated with anti-HIF-1α primary antibody overnight at 4℃, then with fluorescent secondary antibody for 1 h at room temperature, and counterstained with nuclear dye. The subcellular localization of HIF-1α was observed under a fluorescence microscope[1]

Animal/Disease Models: BALB/c athymic nude mice injected with HCT116 cells [1]
Doses: 10 or 30 mg/kg
Route of Administration: po (oral gavage); 10 or 30 mg/kg; once every 3 days; 30-day
Experimental Results: based on dose Dependent manner inhibits the protein levels of HIF-1α, p-STAT-3 (Tyr705), PD-L1 and VEGF in tumor tissues.

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1. Xenograft tumor model establishment: BALB/c nu/nu nude mice (6-8 weeks old, male) were subcutaneously injected with 1×10⁶ HCT116 colon cancer cells suspended in a mixture of PBS and matrix gel (1:1, v/v) into the right flank. Tumors were allowed to grow to a volume of approximately 100 mm³ (about 7 days after inoculation) before grouping for treatment[1]
2. Drug administration protocol: Mice were randomly divided into three groups (vehicle control, 20 mg/kg Panaxadiol, 40 mg/kg Panaxadiol), with 6 mice per group. Panaxadiol was dissolved in DMSO first (stock solution), then diluted with normal saline to the final concentration (DMSO final concentration < 0.1%) before administration. The drug was delivered via intraperitoneal injection at a volume of 10 μL per gram of mouse body weight, once daily for 21 consecutive days. The vehicle group received the same volume of DMSO-saline mixture without Panaxadiol[1]
3. Sample collection and detection: During the administration period, mouse body weight and tumor volume (measured by caliper, volume = length × width² / 2) were recorded every 3 days. After the final administration, mice were euthanized, tumor tissues were dissected and weighed, and a portion of tumor tissue was fixed in formalin for immunohistochemical staining, while another portion was used for protein extraction and western blot analysis[1]

1. In vivo acute toxicity: At therapeutic doses (20 mg/kg and 40 mg/kg, intraperitoneal injection, for 21 days), Panaxadiol did not cause significant weight loss (maximum weight change less than 5% of baseline) or significant pathological damage to major organs (liver, kidney, spleen, lung) in nude mice [1]. 2. In vitro cytotoxicity: Panaxadiol exhibited selective antiproliferative activity against colon cancer cells (HCT116/SW480), and showed extremely low cytotoxicity against normal intestinal epithelial cells (NCM460) at concentrations up to 40 μM (cell survival rate > 85% after 48 hours of treatment) [1].

[1]. Panaxadiol inhibits programmed cell death-ligand 1 expression and tumour proliferation via hypoxia-inducible factor (HIF)-1α and STAT3 in human colon cancer cells. Pharmacol Res. 2020;155:104727.

[2]. Development of natural products for anti-PD-1/PD-L1 immunotherapy against cancer. J Ethnopharmacol. 2021 Dec 5;281:114370.

Panaxadiol is a triterpenoid saponin.
It has been reported that ginseng contains Panaxadiol, and there are related data reports.

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1. Panaxadiol is the main aglycone metabolite of ginsenoside (the active ingredient of ginseng CA Mey.), which plays a dual anti-tumor role in colon cancer: directly inhibiting tumor cell proliferation and indirectly regulating the expression of immune checkpoint (PD-L1) by targeting the HIF-1α and STAT3 signaling pathways [1] 2. The anti-PD-L1 mechanism of Panaxadiol does not depend on direct binding to PD-L1; instead, it inhibits the nuclear translocation and transcriptional activity of HIF-1α and inhibits the phosphorylation of STAT3, thereby reducing the transcription of the PD-L1 gene in tumor cells under hypoxic conditions (a common feature of the solid tumor microenvironment) [1] 3. Panaxadiol is a natural product with the potential for anti-PD-1/PD-L1 cancer immunotherapy; compared with synthetic PD-L1 inhibitors, it has the advantages of low toxicity and natural source, and can be developed as a monotherapy or in combination with existing immune checkpoint inhibitors to improve therapeutic efficacy and reduce side effects [2]

C30H52O3

460.7321

460.391

19666-76-3

73498

White to off-white solid powder

1.0±0.1 g/cm3

531.3±45.0 °C at 760 mmHg

247 °C

275.1±28.7 °C

0.0±3.2 mmHg at 25°C

1.515

7.64

2

3

1

33

789

10

C[C@@]1(CCCC(O1)(C)C)[C@H]2CC[C@@]3([C@@H]2[C@@H](C[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)O)C)C)O)C

PVLHOJXLNBFHDX-XHJPDDKBSA-N

InChI=1S/C30H52O3/c1-25(2)13-9-14-30(8,33-25)19-10-16-29(7)24(19)20(31)18-22-27(5)15-12-23(32)26(3,4)21(27)11-17-28(22,29)6/h19-24,31-32H,9-18H2,1-8H3/t19-,20+,21-,22+,23-,24-,27-,28+,29+,30+/m0/s1

(3S,5R,8R,9R,10R,12R,13R,14R,17S)-4,4,8,10,14-pentamethyl-17-[(2R)-2,6,6-trimethyloxan-2-yl]-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3,12-diol

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 : ~100 mg/mL (~217.05 mM)
Ethanol : ~20 mg/mL (~43.41 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.43 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 (5.43 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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 (Please use freshly prepared in vivo formulations for optimal results.)