YL-109 (chemical structure: 2-(4-Hydroxy-3-methoxyphenyl)-benzothiazole) exerts its antitumor effects by inducing the expression of the ubiquitin ligase CHIP (Carboxyl terminus of Hsc70-interacting protein). [1]

In breast cancer cells, YL-109 (0.001-10 μM; 96 h or 24 h) inhibits cell motility, proliferation, and invasiveness[1]. In MDA-MB-231 cells, YL-109 (1 μM) raises CHIP mRNA and protein levels[1].

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1. Inhibition of breast cancer cell proliferation: YL-109 dose-dependently inhibited the proliferation of human breast cancer cell lines MDA-MB-231 (triple-negative) and MCF-7 (ER-positive) after 24, 48, and 72 hours of treatment (MTT assay). The proliferation inhibition rate increased with increasing drug concentration and treatment time, with significant inhibition observed at concentrations ≥10 μM. [1]

2. Suppression of cell migration and invasion: YL-109 (10, 20 μM) significantly reduced the migration and invasion capabilities of MDA-MB-231 cells (Transwell assay and wound-healing assay). The number of migrated/invasive cells in the YL-109-treated groups was significantly lower than that in the vehicle control group, and the wound closure rate was also significantly decreased. [1]

3. Induction of breast cancer cell apoptosis: YL-109 (20 μM) induced apoptosis in MDA-MB-231 and MCF-7 cells (Annexin V-FITC/PI double staining and flow cytometry). The apoptotic rate was significantly higher than that in the control group, accompanied by upregulation of pro-apoptotic proteins (Bax, Cleaved-Caspase 3, Cleaved-Caspase 9) and downregulation of anti-apoptotic protein Bcl-2 (Western blot analysis). [1]

4. Regulation of CHIP and oncogenic protein expression: YL-109 dose-dependently upregulated CHIP protein and mRNA expression in breast cancer cells (Western blot and real-time PCR). Concurrently, it downregulated the expression of oncogenic proteins including HER2, EGFR, Akt, and STAT3 (Western blot), which are known substrates of CHIP-mediated ubiquitination. [1]

5. Mechanism validation via CHIP knockdown: Transfection of MDA-MB-231 cells with CHIP-specific siRNA significantly attenuated the antitumor effects of YL-109, including reduced proliferation inhibition, migration/invasion suppression, and apoptosis induction. CHIP knockdown also reversed the YL-109-mediated downregulation of HER2, EGFR, Akt, and STAT3, confirming that CHIP induction is the key mechanism of YL-109’s action. [1]

6. Enhancement of CHIP-mediated ubiquitination: YL-109 (20 μM) promoted the ubiquitination of HER2 and EGFR in MDA-MB-231 cells (co-immunoprecipitation assay with anti-ubiquitin antibody), indicating that YL-109 induces CHIP to mediate the ubiquitin-proteasome degradation of oncogenic proteins. [1]

In vivo, YL-109 (15 mg/kg; sc every 2 days) suppresses the growth of tumors as well as the metastasis of breast cancer cells[1].

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1. Inhibition of tumor growth in nude mouse xenograft model: [1]

- Female BALB/c nude mice were subcutaneously inoculated with MDA-MB-231 cells (1×10⁶ cells/mouse) to establish xenograft tumors. When tumors reached a volume of ~100 mm³, YL-109 was administered via intraperitoneal injection at doses of 10 and 20 mg/kg once every 2 days for 3 weeks.
- YL-109 dose-dependently inhibited tumor growth: the final tumor volume and weight in the 10 mg/kg and 20 mg/kg groups were significantly lower than those in the vehicle control group. The 20 mg/kg dose exhibited the most potent antitumor effect, with a tumor growth inhibition rate of ~60%.
- Immunohistochemical (IHC) staining of tumor tissues showed that YL-109 treatment upregulated CHIP expression and downregulated HER2, EGFR, p-Akt, and p-STAT3 expression. TUNEL staining confirmed increased apoptotic cells in tumor tissues from YL-109-treated mice.
2. Suppression of lung metastasis in nude mouse model: [1]

- Female BALB/c nude mice were intravenously injected with MDA-MB-231 cells (5×10⁵ cells/mouse) to induce lung metastasis. YL-109 (20 mg/kg) was administered via intraperitoneal injection once every 2 days for 4 weeks.
- YL-109 significantly reduced the number of lung metastatic nodules compared to the vehicle control group (gross observation and HE staining of lung tissues). IHC staining of lung metastatic lesions showed upregulated CHIP expression and downregulated HER2/EGFR/Akt/STAT3 signaling pathway proteins. [1]

Cell Proliferation Assay[1]
Cell Types: MCF-7 and MDA-MB-231 cells
Tested Concentrations: 0.001, 0.01, 0.1, 1, 10 μM
Incubation Duration: 96 hrs (hours)
Experimental Results: Strongly inhibited cell proliferation of MCF -7 and MDA-MB-231 cells in a dose-dependent manner (IC50=85.8 nM and 4.02 μM, respectively).

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1. Cell proliferation MTT assay: [1]

MDA-MB-231 and MCF-7 cells were seeded in 96-well plates at a density of 5×10³ cells/well and incubated overnight. Cells were treated with serial concentrations of YL-109 (0, 5, 10, 20, 40 μM) for 24, 48, or 72 hours. After treatment, MTT reagent was added to each well and incubated for 4 hours. The supernatant was discarded, and formazan crystals were dissolved in a solubilization solution. The absorbance was measured at 570 nm using a microplate reader, and the cell proliferation inhibition rate was calculated.
2. Cell migration and invasion Transwell assay: [1]

For migration assay: MDA-MB-231 cells were resuspended in serum-free medium containing YL-109 (0, 10, 20 μM) and seeded into the upper chamber of Transwell inserts. Medium containing 10% FBS was added to the lower chamber. After 24 hours of incubation, non-migrated cells on the upper surface of the membrane were removed, and migrated cells on the lower surface were fixed, stained, and counted under a microscope.
For invasion assay: Matrigel-precoated Transwell inserts were used, and the experimental procedure was the same as the migration assay, with incubation extended to 48 hours.
3. Cell apoptosis Annexin V-FITC/PI staining assay: [1]

MDA-MB-231 and MCF-7 cells were treated with YL-109 (20 μM) for 48 hours. Cells were harvested, washed with PBS, and resuspended in binding buffer. Annexin V-FITC and PI were added to the cell suspension and incubated in the dark for 15 minutes. Apoptotic cells were analyzed by flow cytometry, and the apoptotic rate was calculated.
4. Western blot analysis for protein expression: [1]

Cells or tumor tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using a BCA assay. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with non-fat milk. Membranes were incubated with primary antibodies against CHIP, HER2, EGFR, Akt, p-Akt, STAT3, p-STAT3, Bcl-2, Bax, Cleaved-Caspase 3, Cleaved-Caspase 9, or β-actin overnight at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies, and protein bands were visualized using an ECL detection system.
5. CHIP siRNA transfection assay: [1]

MDA-MB-231 cells were seeded in 6-well plates and transfected with CHIP-specific siRNA or non-targeting control siRNA using a transfection reagent. After 48 hours of transfection, cells were treated with YL-109 (20 μM) for 48 hours. Cell proliferation, migration, invasion, and apoptosis were detected using MTT, Transwell, and Annexin V-FITC/PI assays, respectively. Western blot was used to verify CHIP knockdown efficiency and the expression of downstream proteins.
6. Co-immunoprecipitation (Co-IP) assay for ubiquitination: [1]

MDA-MB-231 cells were treated with YL-109 (20 μM) for 24 hours, and a proteasome inhibitor was added 6 hours before harvest. Cells were lysed in IP buffer, and the lysate was incubated with anti-HER2 or anti-EGFR antibody overnight at 4°C. Protein A/G agarose beads were added and incubated for 4 hours. Beads were washed, and immunoprecipitated complexes were eluted and subjected to Western blot analysis using anti-ubiquitin antibody to detect ubiquitinated HER2/EGFR. [1]

Animal/Disease Models: BALB/cAjcl-nu/nu female mice (4-5 weeks) inoculated with MCF-7 or MDA-MB-231 cells[1]
Doses: 15 mg/kg
Route of Administration: Sc every 2 days for 63 days
Experimental Results: Suppressed tumor growth in mice injected with MCF-7 and MDA-MB-231 cells.

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1. Tumor xenograft model in nude mice: [1]

- Animals: Female BALB/c nude mice (6-8 weeks old) were housed under specific pathogen-free (SPF) conditions.
- Tumor inoculation: MDA-MB-231 cells (1×10⁶ cells in 100 μL PBS) were subcutaneously injected into the right flank of each mouse.
- Grouping and drug administration: When tumors grew to ~100 mm³, mice were randomly divided into three groups (n=6 per group): vehicle control group, YL-109 10 mg/kg group, and YL-109 20 mg/kg group. YL-109 was dissolved in a suitable vehicle (e.g., DMSO:PEG400:PBS = 1:4:5) and administered via intraperitoneal injection once every 2 days for 3 weeks. The vehicle control group received the same volume of vehicle.
- Tumor measurement: Tumor volume was measured every 3 days using a caliper, calculated as V = (length × width²)/2.
- Sample collection: After 3 weeks of treatment, mice were euthanized. Tumors were excised, weighed, and fixed in formalin for IHC staining.
- IHC detection: Formalin-fixed tumor tissues were embedded in paraffin, sectioned, and stained with antibodies against CHIP, HER2, EGFR, p-Akt, p-STAT3, and Ki-67. TUNEL staining was performed to detect apoptotic cells.
2. Lung metastasis model in nude mice: [1]

- Animals: Female BALB/c nude mice (6-8 weeks old) were used.
- Metastasis induction: MDA-MB-231 cells (5×10⁵ cells in 100 μL PBS) were intravenously injected via the tail vein.
- Drug administration: One day after cell injection, mice were randomly divided into two groups (n=6 per group): vehicle control group and YL-109 20 mg/kg group. YL-109 was administered via intraperitoneal injection once every 2 days for 4 weeks, with the same vehicle as the xenograft model.
- Sample collection: After 4 weeks of treatment, mice were euthanized. Lungs were excised, fixed in formalin, and subjected to gross observation and HE staining to count metastatic nodules. IHC staining of lung tissues was performed to detect CHIP, HER2, and EGFR expression. [1]

[1]. 2-(4-Hydroxy-3-methoxyphenyl)-benzothiazole suppresses tumor progression and metastatic potential of breast cancer cells by inducing ubiquitin ligase CHIP. Sci Rep. 2014 Nov 18;4:7095.

1. Chemical structure: The chemical structure of YL-109 is 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole, belonging to the benzothiazole derivative family. [1] 2. Background: Breast cancer is one of the most common malignant tumors in women, and metastatic breast cancer has a poor prognosis. Oncogenes such as HER2, EGFR, Akt and STAT3 are overexpressed in breast cancer, promoting tumor progression and metastasis. CHIP is a ubiquitin ligase that mediates the ubiquitin-proteasome degradation of oncogenes, and its downregulation is associated with breast cancer progression. [1] 3. Mechanism of action: YL-109 exerts its antitumor effect by specifically inducing CHIP expression. Inducible CHIP mediates the ubiquitination and degradation of HER2, EGFR, Akt and STAT3, thereby inhibiting the proliferation, migration and invasion of breast cancer cells and inducing apoptosis. [1]
4. Therapeutic Potential: YL-109 has shown potent antitumor and antimetastatic activity in preclinical breast cancer models, particularly in triple-negative breast cancer (MDA-MB-231) lacking targeted therapy. It offers a potential therapeutic strategy for breast cancer by targeting the CHIP oncoprotein pathway. [1]

C14H11NO2S

257.31

257.051

36341-25-0

3155228

Light yellow to yellow solid powder

1.327g/cm3

446.448ºC at 760 mmHg

223.804ºC

1.685

3.677

1

4

2

18

289

0

KRVBOHJNAFQFPW-UHFFFAOYSA-N

InChI=1S/C14H11NO2S/c1-17-12-8-9(6-7-11(12)16)14-15-10-4-2-3-5-13(10)18-14/h2-8,16H,1H3

4-(1,3-benzothiazol-2-yl)-2-methoxyphenol

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)

Solubility in Formulation 1: ≥ 3 mg/mL (11.66 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 30.0 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: 3 mg/mL (11.66 mM) 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; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 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: ≥ 3 mg/mL (11.66 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 30.0 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.)