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Purity: ≥98%
Chetomin (also called NSC289491, BRN0077366), an antibiotic metabolite of chaetomium cochliodes and a natural product isolated from Chaetomium species which has anticancer, antibacterial and antifungal properties, is an inhibitor of HIF-1 which weakens transcription of HIF-1, and disrupts the binding of HIF-1α and HIF-2α to p300 at low nanomolar concentrations. Chetomin may find application as a chemotherapeutic agent for cancer treatment. Chetomin specifically inhibits HIF-1 activities by interfering with HIF-1's interaction with p300, a transcriptional coactivator. In vitro, chetomin-induced HIF-1 inhibition significantly lowers hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells. Additionally, chemomin causes XIAP to break down and increases TRAIL sensitivity in urogenital cancer cells.
| Targets |
HSP90; Hsp90/HIF1α
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| ln Vitro |
Hypoxia-inducible factors 1 (HIF-1) is a transcription factor that reacts to oxygen changes in the cellular environment. Chetomin specifically inhibits HIF-1 activities by interfering with HIF-1's interaction with p300, a transcriptional coactivator. In vitro, chetomin-induced HIF-1 inhibition significantly lowers hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells. Chetomin reduces malignant glioma cell lines U251MG (DMF10: 1.35 and 1.18) and U343MG (DMF10: 1.78 and 1.48) radioresistance caused by hypoxia. Hypoxia-inducible factor-1α's differentiation-inhibitory effect is eliminated when HIF-1 is targeted with chetomin.
Chetomin is an inhibitor of Hsp90/HIF1α activity [1] Chetomin is found in the methanol extract of C. globosum and has been previously demonstrated to inhibit HIF1α binding to other key protein moieties. Therefore, its ability to block Hsp90/HIF1α pathway activity was evaluated in the NSCLC cell lines H1299 and H460. In the following experiments, micromolar levels of chetomin were used for monolayer experiments, while nanomolar levels of chetomin were employed for spheroid cultures of NSCLC cell lines due to differences in IC50 levels (Supplementary Tables 1 and 2). Chetomin exerts its inhibitory action on NSCLC CSCs by eliciting apoptosis [1] We had a particular interest in targeting CSCs as a strategy for overcoming tumor recurrence; therefore, we next tested whether chetomin could target CSCs, initially testing chetomin on adhered monolayer cultures. Chetomin exposure for 24 hours dose-dependently lowered expression of stem cell biomarkers, SRY (sex determining region Y)-box 2 (Sox2), homeobox protein Nanog, and octamer-binding transcription factor 4 (Oct4), in H1299 monolayer cultures (Figure 2A). Additionally, chetomin pretreatment of monolayered H460 and H1299 cells for 24 hours attenuated their sphere formation capacity and number of cells per sphere (Figure 2B). Chetomin exerts its inhibitory action on NSCLC non-CSCs by eliciting apoptosis [1] The majority of tumor cells are non-CSCs, so eliminating them constitutes an important aspect of cancer treatment. Therefore, we next assessed chetomin’s effects on NSCLC non-CSCs. Activating KRAS and inactivating TP53 mutations are frequently recurring features of NSCLC lung cancer cells and CSCs. Hence, we compared the efficacy of chetomin on immortalized human bronchial epithelial (hBE) cells against hBE cells with: (i) a constitutively active KRASG12V mutation (KRASG12V-hBE), (ii) TP53 deficient via a short interfering RNA (siRNA) (siTP53-hBE), or (iii) both mutant KRASG12V and TP53 deficient (KRAS;G12V siTP53-hBE) (Supplementary Figure 2). Chetomin exposure for three days did not significantly inhibit proliferation of hBE cells but did adversely affect the proliferation of KRASG12V-hBE, siTP53-hBE, and KRAS;G12V siTP53-hBE cells (Figure 3A; Supplementary Table 2) and of the diverse NSCLC cell lines and the two paclitaxel-resistant sublines (H460/R and H226B/R; Figure 3B; Supplementary Table 2). The IC50s against the NSCLCs were in the micromolar range, almost a hundred times less potent than the inhibitory action of chetomin on sphere formation, which was in the nanomolar range. Therefore, while chetomin demonstrates some efficacy against non-CSCs, it is more potent against CSCs. Chetomin blocks Hsp90’s binding to HIF1α’s N-terminus [1] We explored the molecular details of the inhibitory action of chetomin on Hsp90/HIF1α binding. We confirmed that Chetomin did not affect Hsp90 or Hsp70 protein levels in 24 hour-exposed monolayer NSCLC cultures (Figure 5A). Since chetomin did not impact Hsp90 protein levels, we next sought whether it directly inhibits the interaction between Hsp90 and HIF1α. It has been well-established that the Hsp90 protein binds to HIF1α’s basic helix–loop–helix–PER–ARNT–SIM (bHLH–PAS) domain, which is located at HIF1α’s N-terminus. Chetomin was therefore titrated into full-length (“FL”), N-terminus domain (“N”), and C-terminus domain (“C”) variants of HIF1α in the presence of Hsp90. Addition of chetomin inhibited the interaction of Hsp90 to the “FL” and “N” variants but not to the “C” variant (Figure 5B), suggesting chetomin’s specifically inhibits the Hsp90-HIF1α binding interaction in HIF1α’s N-terminus. We generated stable lines of H1299 cells with short hairpin RNA (shRNA)-mediated knockdown of Hsp90 (Hsp90-shRNA-H1299) and scrambled control (scr-shRNA-H1299) to determine whether Chetomin’s anti-cancer properties are associated with its inhibition of Hsp90’s binding to HIF1α. As expected, chetomin exposure for two weeks lowered colony- and sphere-forming capacity in scr-shRNA-H1299 cells (Figure 5C, D). However, chetomin exposure did not exert any discernible impact on these characteristics in Hsp90-shRNA-H1299 cultures (Figure 5C, D). Furthermore, chetomin did not affect levels of Akt, an Hsp90 substrate, in Hsp90-shRNA-H1299 cultures (Figure 5E). On the other hand, H1299 cultures overexpressing Hsp90 reestablished levels of Hsp90 substrates MEK1/2 and Src (Figure 5F). These results suggest that chetomin’s anti-cancer properties are dependent upon the presence of Hsp90. |
| ln Vivo |
In an in vivo xenograft model, the coadministration of Chetomin and forskolin significantly suppresses the growth of malignant gliomas. Che-M (chetomin loaded micelles) significantly suppresses zebrafish tumor growth, tumor-induced angiogenesis, and embryonic angiogenesis. Che-M inhibits the growth of tumors in mice and increases the survival time of the subcutaneous CT26 tumor model.
Chetomin inhibits lung tumorigenesis in NSCLC mouse models [1] To corroborate our in vitro findings, we next tested chetomin in vivo in several mouse models. In the KrasLA1 mouse model of spontaneous lung tumorigenesis,25 administration of chetomin for eight weeks strikingly lowered the number of lesions compared to vehicle (Figure 4A). Microexamination of lung tissue revealed dramatically lower lesion counts, size, and total burden in chetomin- versus vehicle-administered groups (Figure 4B). Fluorescence immunohistochemistry (IHC) of lung sections revealed dysregulation of Cl-Cas3, HIF1α, CD34, and Oct4 expression in chetomin- versus vehicle-administered groups (Figure 4C, D). Chetomin exposure did not produce any observable variation in body weight in KrasLA1 mice (Supplementary Figure 4a). The second model we tested Chetomin in vivo on was an H1299 flank xenograft model. When xenografts attained a size ranging from 50 to 150 mm3, mice were randomized to chetomin (50 or 100 mg/kg, p.o.) or vehicle for three weeks. At both doses, chetomin markedly lowered xenograft tumor size (Figure 4E) and mass (Figure 4F). Fluorescence IHC of lung sections revealed a higher intensity of Cl-Cas3 (more apoptosis) and lower intensities of HIF1α and CD34 (less angiogenesis) in chetomin- versus vehicle-administered groups (Figure 4G). Chetomin exposure did not produce any observable variation in body weight in H1299 xenograft mice (Supplementary Figure 4b). Due to their ability to self-renew, CSCs can propagate tumors when inoculated into mice; therefore, we also tested the impact of Chetomin on tumor propagation of CSCs. First, H1299 flank xenografts were established and mice received chetomin or vehicle as described above. The tumors were excised, dissociated, and serially-diluted cells were implanted into recipient NOD/SCID animals. As observed in vitro (Figure 2E), recipients inoculated with chetomin-exposed cells exhibited drastically lower tumorigenesis compared to recipients of vehicle-exposed cells (Figure 4H). Finally, we comparatively evaluated the effects of Chetomin in vivo on a H460 flank xenograft model versus a H460/R flank xenograft model in order to test whether chetomin has a differential effect on chemoresistant H460/R xenografts. When xenografts attained a size ranging from 50 to 150 mm3, mice were randomized to chetomin (50 or 100 mg/kg, p.o.) or vehicle for three weeks. At both doses, H460/R xenograft tumor size (Figure 4I) and mass (Figure 4J) were not significantly different than those of H460 xenografts. Moreover, fluorescence IHC of lung sections revealed no significant differences in Cl-Cas3 (apoptosis) or HIF1α and CD34 (angiogenesis) between chetomin-treated H460/R xenografts versus chetomin-treated H460 xenografts (Figure 4K). Chetomin exposure did not produce any observable variation in body weight in either H460 xenograft mice or H460/R xenograft mice (Supplementary Figure 4C, D). |
| Cell Assay |
Chetomin is added to fully supplemented medium in RT-PCR and clonogenic survival experiments at a concentration of 150 nM four hours prior to treatment with hypoxia. After that, without altering the medium, HT 1080 cells are moved to either the well-humidified incubator (12 hours) or the hypoxic workstation (0.1% O2). Therefore, HT1080 cells are treated with chetomin (150 nM) for 16 hours before receiving radiation therapy.
Quantification of proliferation by MTT assay [1] 5 × 103 cells per well were plated onto 96-well culture plates and provided 24 hours to adhere. Methanol extract, Chetomin, or vehicle was then added to the cell culture media at specified concentrations and incubated for three days (37°C, 5% CO2). At this stage, MTT was added to the culture media to a final amount of 500 μg/ml and culture plates were returned to the 37°C incubator for 2 to 4 hours. Formazan produced by cells still living was solubilized in DMSO and the colorimetric signal quantified at 570 nm. All experimental results are relative to the control value and expressed as a percent. Assessment of CSC-like characteristics by sphere-forming assay [1] Cells were grown in media to sustain sphere formation in ultra-low attachment 96-well culture plates. The culturing media consisted of DMEM-F12 with B-27™ supplement, growth factors (bFGF, EGF), and antibiotics. Spheres were incubated with Chetomin or vehicle at the specified concentrations in an incubator (37°C, 5% CO2) for two weeks or till spheres were larger than 150 µm3. Assessment of colony-forming capacity by an anchorage-dependent assay [1] 300 cells per well were plated to 6-well culture plates and Chetomin or vehicle with/without caspase-3 inhibitor was added at the specified concentrations and incubated for two weeks (37°C, 5% CO2). Fresh culture media was replenished 1 or 2 times per week. At the end of the incubation period, fixation of colonies was performed with methanol (100%), followed by staining with crystal violet (0.002% in water), and by multiple rinse steps using deionized water. Colonies were visualized and then quantified by ImageJ. Assessment of colony-forming capacity by an anchorage-independent (soft agar) assay [1] Cells were suspended in sterile agar (1%), resulting in a 0.4% agar solution, which was added into the wells of a 24-well culture plate that had been pre-coated with agar (1%). Culture media with Chetomin or vehicle with/without caspase-3 inhibitor at the specified concentrations was overlaid over the solidified agar and incubated for two weeks (37°C, 5% CO2). Fresh culture media was replenished 1 or 2 times per week. At the end of the incubation period, staining of live colonies was performed by MTT solution, which were then visualized, and then quantified by ImageJ. Evaluation of apoptosis by cell-cycle analysis [1] Cells were cultured with varying levels of Chetomin or vehicle for 24 hours. Both suspended and adherent cells were harvested, rinsed with phosphate buffered saline (PBS), and fixation was performed with methanol (100%). Staining was achieved with PI supplemented with RNase A (both at 50 μg/ml) for 30 minutes, room temperature (RT). Readout was performed by flow cytometry on a BD FACSCalibur®; a control sample was used for gating and fluorescence intensity was measured. BD CellQuest™ was employed to analyze cell-cycle progression. Quantification of endothelial cell tube formation Preparation of conditioned media (CM): H1299 cells were cultured with Chetomin or vehicle for 24 hours, followed by an additional incubation period of 4 hours in a hypoxic (1% O2) or normoxic (room air, 21% O2) environment. At this point, the media containing chetomin or vehicle was replaced by new, serum-deficient culture media and incubated an additional 24 hours. The resultant CM was collected for subsequent experiments. Tube formation assay: [1] The procedure adopted for assessing tube-forming capacity was executed according to published reports.18 In brief, HUVECs were plated into the wells of CellBIND® 96-well culture plates and given time to adhere. They were then treated with regular media or CM (normoxic or hypoxic) from Chetomin- or vehicle-treated H1299 cultures. HUVECs were visualized, and alterations in morphology were scored. Analysis of NSCLC culture protein and mRNA content [1] Cell cultures were incubated with specified concentrations of Chetomin or vehicle for 24 hours. For protein analyses, cell lysates were subjected to Western blotting as described in the Supplementary Information. For mRNA analyses, a RNA extraction kit was used to purify total RNA from cells. Reverse transcription PCR (RT-PCR) was conducted according to published methods19 using primer pairs outlined in Supplementary Table 3. The amplified DNA was resolved by agarose gel (1.5%) electrophoresis and imaged with a Gel Doc™ EZ System. Pull-down, immunoprecipitation (IP), and competitive ATP-binding assays [1] Pull-down, IP assays, and purification of Hsp90 with Chetomin or vehicle were conducted according to published reports. Interaction between Hsp90 and Chetomin by fluorescence titration [1] Fluorescence titration experiments were conducted in order to evaluate the interaction between Hsp90 and chetomin, and to identify the possible binding location(s) of chetomin to Hsp90, as described in the Supplementary Information. |
| Animal Protocol |
Chetomin was loaded in micelles; 1 mg/mL(for zebrafish); 2 mg/kg(for mouse); s.c.
Transgenic fish Tg(flk1:EGFP); Mouse model(BALB/c mice) Tumor-propagation from dissociated tumor xenografts [1] Following the 8-week regimen of Chetomin or vehicle, mice were sacrificed, tumors excised, and dissociated using a Tumor Dissociation Kit. Viable individual cancer tells were counted with trypan blue and 500 to 50,000 cells were implanted in NOD/SCID animals by subcutaneous injection in their right flank. Mice were monitored, and tumor burden assessed. |
| Toxicity/Toxicokinetics |
rat LD50 oral 75 mg/kg CRC Handbook of Antibiotic Compounds, Vols.1- , Berdy, J., Boca Raton, FL, CRC Press, 1980, 4(1)(174), 1980
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| References | |
| Additional Infomation |
Chetomin has been reported in Chaetomium cochliodes, Chaetomium elatum, and other organisms with data available.
Non-small cell lung cancer (NSCLC) remains recalcitrant to effective treatment due to tumor relapse and acquired resistance. Cancer stem cells (CSCs) are believed to be one mechanism for relapse and resistance and are consequently considered promising drug targets. We report that chetomin, an active component of Chaetomium globosum, blocks heat shock protein 90/hypoxia-inducible factor 1 alpha (Hsp90/HIF1α) pathway activity. Chetomin also attenuated sphere-forming, a stem cell-like characteristic, of NSCLC CSCs (at ~ nM range) and the proliferation of non-CSCs NSCLC cultures and chemoresistant sublines (at ~ μM range). At these concentrations, chetomin exerted a marginal influence on noncancerous cells originating from several organs. Chetomin markedly decreased in vivo tumor formation in a spontaneous KrasLA1 lung cancer model, flank xenograft models, and a tumor propagation flank implanted model at doses that did not produce an observable toxicity to the animals. Chetomin blocked Hsp90/HIF1α pathway activity via inhibiting the Hsp90-HIF1α binding interaction without affecting Hsp90 or Hsp70 protein levels. This study advocates chetomin as a Hsp90/HIF1α pathway inhibitor and a potent, nontoxic NSCLC CSC-targeting molecule. [1] Herein, we report that Chetomin, an indole heteropentacyclic compound isolated from C. globosum, functions as an Hsp90/HIF1α pathway inhibitor that decreases the viability and elicits apoptosis of NSCLC CSCs spheres and non-CSCs monolayer cultures in vitro within nanomolar and micromolar range, respectively. Moreover, we found that chetomin inhibits tumorigenesis in a spontaneous KrasLA1 lung cancer model, a H1299 flank xenograft model, and a tumor propagation flank implanted model. Notably, chetomin does not significantly affect Hsp90 or Hsp70 expression in vitro, is only marginally toxic to non-cancerous cell lines in vitro, and produces no discernible toxicity to mice in vivo. These properties make chetomin particularly attractive as a potential anti-cancer drug in NSCLC patients. On a molecular level, Chetomin has been shown to target the CH1 domain of the transcriptional co-activator p300, thereby blocking the p300-HIF-1α interaction, which attenuates HIF-1α’s hypoxia-inducible transcription. As we found that chetomin blocks Hsp90’s binding to HIF1α, these findings suggest that chetomin indirectly produces this effect through p300. On a cellular level, chetomin inhibits human triple-negative breast cancer cell proliferation via apoptosis induction and displays anti-myelomatous activity without toxicity to normal hematopoietic cells This evidence supports our contention that chetomin displays potent anti-cancer properties with a favorable safety profile. In conclusion, our study advocates chetomin as a Hsp90/HIF1α pathway inhibitor and a potent, nontoxic NSCLC CSC-targeting molecule. |
| Molecular Formula |
C31H30N6O6S4
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| Molecular Weight |
710.87
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| Exact Mass |
712.126
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| Elemental Analysis |
C, 52.38; H, 4.25; N, 11.82; O, 13.50; S, 18.04
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| CAS # |
1403-36-7
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| Related CAS # |
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| PubChem CID |
10417379
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| Appearance |
White to light yellow solid powder
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| Density |
1.8±0.1 g/cm3
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| Melting Point |
710.9ºC
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| Index of Refraction |
1.869
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| LogP |
3.85
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
47
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| Complexity |
1460
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| Defined Atom Stereocenter Count |
6
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| SMILES |
CN1C(=O)[C@@]2(CO)N(C)C(=O)[C@]1(CC3=CN(C4=CC=CC=C34)[C@]56C[C@]78C(=O)N(C)[C@](CO)(C(=O)N7[C@H]5NC9=CC=CC=C96)SS8)SS2
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| InChi Key |
ZRZWBWPDBOVIGQ-YWZWRZHGSA-N
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| InChi Code |
InChI=1S/C31H30N6O6S4/c1-33-25(42)30(15-38)34(2)23(40)28(33,44-46-30)12-17-13-36(21-11-7-4-8-18(17)21)27-14-29-24(41)35(3)31(16-39,47-45-29)26(43)37(29)22(27)32-20-10-6-5-9-19(20)27/h4-11,13,22,32,38-39H,12,14-16H2,1-3H3/t22-,27+,28+,29+,30+,31+/m1/s1
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| Chemical Name |
(1S,3S,11R,14S)-14-(hydroxymethyl)-3-[3-[[(1S,4S)-4-(hydroxymethyl)-5,7-dimethyl-6,8-dioxo-2,3-dithia-5,7-diazabicyclo[2.2.2]octan-1-yl]methyl]indol-1-yl]-18-methyl-15,16-dithia-10,12,18-triazapentacyclo[12.2.2.01,12.03,11.04,9]octadeca-4,6,8-triene-13,17-dione
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4067 mL | 7.0336 mL | 14.0673 mL | |
| 5 mM | 0.2813 mL | 1.4067 mL | 2.8135 mL | |
| 10 mM | 0.1407 mL | 0.7034 mL | 1.4067 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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