http://orcid.org/0000-0003-2988-3786
Figures
Abstract
The JAK2/STAT3 signaling pathway plays a critical role in tumorigenesis, and has been suggested as a potential molecular target for anti-melanoma therapeutics. However, few JAK2 inhibitors were being tested for melanoma therapy. In this study, eight amentoflavone analogues were evaluated for their activity against human malignant melanoma cells. The most potent analogue, compound 1, inhibited the phosphorylation of JAK2 and STAT3 in human melanoma cells, but had no discernible effect on total JAK2 and STAT3 levels. A cellular thermal shift assay was performed to identify that JAK2 is engaged by 1 in cell lysates. Moreover, compound 1 showed higher antiproliferative activity against human melanoma A375 cells compared to a panel of cancer and normal cell lines. Compound 1 also activated caspase-3 and cleaved PARP, which are markers of apoptosis, and suppressed the anti-apoptotic Bcl-2 level. Finally, compound 1 induced apoptosis in 80% of treated melanoma cells. To our knowledge, compound 1 is the first amentoflavone-based JAK2 inhibitor to be investigated for use as an anti-melanoma agent.
Citation: Wu K-J, Huang J-M, Zhong H-J, Dong Z-Z, Vellaisamy K, Lu J-J, et al. (2017) A natural product-like JAK2/STAT3 inhibitor induces apoptosis of malignant melanoma cells. PLoS ONE 12(6): e0177123. https://doi.org/10.1371/journal.pone.0177123
Editor: Salvatore V. Pizzo, Duke University School of Medicine, UNITED STATES
Received: November 18, 2016; Accepted: April 21, 2017; Published: June 1, 2017
Copyright: © 2017 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This work is supported by Hong Kong Baptist University (FRG2/15-16/002), the Health and Medical Research Fund (HMRF/14130522), the Research Grants Council (HKBU/12301115, HKBU/204612 and HKBU/201913), the French National Research Agency/Research Grants Council Joint Research Scheme (A-HKBU201/12 – Oligoswitch), National Natural Science Foundation of China (21575121), Guangdong Province Natural Science Foundation (2015A030313816), Hong Kong Baptist University Century Club Sponsorship Scheme 2016, Interdisciplinary Research Matching Scheme (RC-IRMS/15-16/03), the Science and Technology Development Fund, Macao SAR (098/2014/A2), the University of Macau (MYRG2015-00137-ICMS-QRCM, MYRG2016-00151-ICMS-QRCM and MRG044/LCH/2015/ICMS) to CHL, and National Natural Science Foundation of China (21628502).
Competing interests: The authors have declared that no competing interests exist.
Introduction
The incidence of melanoma has increased over the past three decades [1,2], and its mortality rate is higher than another cancers [3,4]. However, less special drug for metastatic melanoma is approved for the first-line therapy [5–9].The Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway is overactivated in many human cancers, including melanoma [10,11]. Therefore, inhibiting JAK2 is a potential anticancer strategy. AG490, the first JAK2 inhibitor, selectively blocks cell growth in vitro and in vivo by inhibition of JAK2 activity and inducing apoptosis [12]. Another potent JAK2 inhibitor, NVP-BBT594, suppressed activation loop phosphorylation of JAK2 [13]. To the best of our knowledge, just a small number of JAK2 inhibitors were being tested for cancer therapy on the status phase Ⅰ or phase Ⅱ, and only one JAK2 inhibitor, Ruxolitinib (INC424), was approved by Food and Drug Administration (FDA) [14]. Within the realm of natural products or natural product analogues, cucurbitacin Ι and BBMD3 have been reported to inhibit the JAK2/STAT3 pathway [10,15].
Traditional Chinese herbal medicines provide a rich resource of bioactive structure for pharmaceutical drug development [16–36]. Flavonoids are natural polyphenolic substances that are widely found in Traditional Chinese medicine [37] and have been investigated as potential anti-cancer agents [38]. Previously, our group reported that the activities of amentoflavone and its analogues on JAK2 kinase against human erytholeukemia cells (HEL). The biflavonoid amentoflavone was identified as an inhibitor of JAK2 activity using a structure-based virtual screening approach, which showed promising anticancer activity against HEL cells [34]. However, the efficacy of amentoflavone analogues against malignant melanoma, a widespread and deadly cancer, has not yet been investigated. Therefore, in this study, eight amentoflavone analogues were evaluated for their anticancer activities against human melanoma cells. Our findings demonstrate that the amentoflavone analogue compound 1 is a potent inhibitor of the JAK2/STAT3 signaling pathway against melanoma cells, suggesting that this natural product scaffold could deserve further attention for the development of anti-melanoma therapeutics.
Materials and methods
Reagents
All antibodies were purchased from Cell signaling Technology. All compounds were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM. Human malignant melanoma (A375) cells, human malignant melanoma (A2058) cells, human prostate cancer (PC3) cells, human prostate cancer (DU145) cells, and human liver cancer (HepG2) cells were obtained from American Type Culture Collection. The hepatocyte cell line LO2 was obtained from Chinese Academy of Science, Cell Biology of Shanghai Institute, Shanghai, China.
Cell culture
A375, A2058, PC3, DU145, HepG2 and LO2 cells were cultured at cell density of 4–5 × 105 cells/mL in DMEM (Dulbecco’s Modified Eagle Medium) with high glucose and L-glutamine and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin (100 units/mL)/streptomycin (100 μg/mL) at 37°C and 5% CO2.
Cell viability assay
A MTT assay was used to evaluate the antiproliferative effect of the natural products. Cells were seeded at a density of 5,000–6,000 cells per well in 96-well plates. When the density of the cells reached 50% confluence, the cells were treated with compounds at final concentrations ranging from 0.01 to 10 μM for 48 h. MTT was added to each well at a final concentration of 1 mg/mL for a further 4 h. After removing the medium from the cells, 100 μL DMSO was added to each well. The viability of the cells was measured by recording the absorbance of each well at 490 nm using a SpectraMax M5 microplate reader after shaking the plate for 10 min at room temperature in the dark.
Western blotting analysis
A375 cells were treated with vehicle, compound 1 or NVP-BBT594 for 24 h, and then harvested and washed twice with ice-cold PBS. Protein samples were extracted with radio-immunoprecipitation assay buffer (RIPA) lysis buffer containing 1% cocktail and 1% PMSF. 30 μg of total proteins were resolved on an SDS/PAGE gel and transferred to a polvinylidene fluoride (PVDF) membrane. Blots were blocked in 5% none-fat dry milk with TBS containing 0.1% Tween-20 for 1 h and probed with primary antibodies to JAK2, p-JAK2, STAT3, p-STAT3, caspase-3, PARP, Bcl-2, HIF1α, ubiquitin or β-actin and GAPDH with gentle agitation overnight at 4°C. Then, membranes were washed five time with TBST. After incubation with secondary antibody for 1 h. Signals of proteins bands were detected using enhanced chemiluminescent Plus reagents (GE Healthcare) and analyzed by Image Lab.
Cellular thermal shift assay
Cellular thermal shift assay was performed to evaluate the target engagement of compound 1 in A375 cells. Briefly, after collecting A375 cell lysates, the cells lysates was separated in the same aliquots. Each aliquot was treated with 1 (1 μM) or DMSO. After incubation at room temperature for 30 min, the complex-treated lysates were divided into 50 μL in each of PCR tubes and heated individually at different temperatures (Veriti thermal cycler, Applied Biosystems/Life Technologies). The heated lysates were centrifuged and the supernatants were analyzed by SDS-PAGE followed by western-blot assay by probing with anti-JAK2 antibody.
Apoptosis assay
To evaluate apoptosis induction, a FITC-Annexin apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was utilized. The experiment was performed according to the manufacturer’s instructions. Briefly, A375 cells were seeded at density of 2.5 × 105 in a 6-well plate, and were treated with compound 1 at different concentrations (0.03 to 3 μM) for 24 h. Cells were harvested and washed twice with ice-cold PBS, and then resuspended in 1 × binding buffer followed by incubation with Annexin V/PI solution for 15 min at room temperature. The samples were immediately analyzed by flow cytometry using a C6 Flow Cytometer™ system (BD Biosciences). At least 2 × 105 cells were analyzed for each sample.
Results
Identification of 1 as a potent inhibitor of human melanoma cell viability
Eight amentoflavone analogues (Fig 1) were synthesized previously [34]. For primary screening, the MTT assay was performed to assess the ability of the analogues to inhibit the proliferation of A375 cells. Amentoflavone was used as the positive control. The results showed that compound 1 showed potent anticancer activity against the A375 cell line, with an IC50 value of 0.37 μM, compared to other 0.50 μM for amentoflavone (Fig 2A). In addition, compound 1 showed relatively non-toxic towards DU145, PC3, HepG2, A2058 and LO2 cells (IC50 ≥ 10 μM) (Fig 2B–2F). These data suggest that compound 1 could be regarded as a potential chemotherapeutic agent against human melanoma.
A375 cells were treated with 0.01 to 4 μM of compounds or amentoflavone for 48 h. LO2 cells were treated with the same concentration of compound 1 for 48 h. PC3, DU145, HepG2 and A2058 cells were treated with 0.01 to 10 μM of compound 1 for 48 h. Error bars represent the standard deviations of results obtained from three independent experiments.
Effect of compound 1 on the JAK2/STAT3 pathway
In order to determine whether compound 1 inhibits the JAK2/STAT3 pathway in cellulo, Western blotting assays were performed. A375 cells were treated with compound 1 (0.03 to 3 μM) for 24 h before measurement. The results showed that compound 1 suppressed the phosphorylation of JAK2 and STAT3, but had no discernible effect on total JAK2 and STAT3 levels (Fig 3).
A375 cells were treated with the indicated concentrations of compound 1 (0.03 to 3 μM) or positive control compound NVP-BBT594 (1 μM) for 24 h. Protein lysates were analyzed by Western blotting with the indicated antibodies. Error bars represent the standard deviation of triplicate results.
Cellular thermal shift assay
To evaluate whether JAK2 is engaged by 1 in cell lysates, a cellular thermal shift assay (CETSA) was applied [39]. The JAK2 content in the soluble fraction was determined by Western blotting. The results showed that 1 could stabilize JAK2 in treated cell lysates, as indicated by an obvious shift of the melting temperature of JAK2 content in the soluble fraction (Fig 4A and 4B). This result indicates that the thermal stabilization of JAK2 is engaged by 1.
(A) Stabilization of JAK2 by 1. (B) The band intensity of JAK2 with different temperatures. The data were normalized to the JAK2 level of control group at 55°C and are expressed as the means ± SD of three individual experiments. The data were analyzed using Image Lab. Error bars represent the standard deviation of triplicate results. Significant differences versus control at the same temperature are indicated by **p < 0.01.
Compound 1 induces cell apoptosis
Activation of STAT3 can suppress apoptosis through inhibition of activated caspase-3 [40]. The cleavage of PARP and activation of caspase-3 are widely regarded as markers of apoptosis [40]. To confirm whether compound 1 induces apoptosis, A375 cells were exposed to 0.03 to 3 μM of compound 1 for 24 h, and the level of apoptosis was assessed by flow cytometry using annexin V and PI staining. The results showed that compound 1 could induce apoptosis in approximately 80% of the cells (Fig 5A). Moreover, compound 1 induced the cleavage of PARP and decreased caspase-3 levels (Fig 5B). Stat3 is known to regulate the expression of various anti-apoptotic and pro-apoptotic proteins [40]. Our results showed that compound 1 suppressed Bcl-2, an anti-apoptotic protein that is upregulated by STAT3 (Fig 6A and 6B). Taken together, these results indicate that 1 could induce apoptosis in A375 cells, presumably as a result of its effects on PARP, caspase-3 and Bcl-2 that are under the control of the JAK2/STAT3 signaling pathway. Additionally, compound 1 had no effect on HIF1α and ubiquitin levels (Fig 7).
A375 cells were treated with the indicated concentrations of compound 1 (0.03 to 3 μM) or positive control compound NVP-BBT594 (1 μM) for 24 h. (A) A375 cells were stained with PI and Annexin V, and were analyzed by flow cytometry. (B) Protein lysates were analyzed by Western blotting with the indicated antibodies.
(A) A375 cells were treated with the indicated concentrations of compound 1 (0.1 to 3 μM) or positive control compound NVP-BBT594 (1 μM) for 24 h. Protein lysates were analyzed by Western blotting with the indicated antibodies. (B) The data were analyzed using Image Lab. Error bars represent the standard deviation of triplicate results.
A375 cells were treated with the indicated concentrations of compound 1 (0.03 to 3 μM) or positive control compound NVP-BBT594 (1 μM) for 24 h. Protein lysates were analysed by Western blotting with the indicated antibodies.
Discussion
STAT3 signaling plays a crucial role in tumorigenesis. In particular, JAK2 is important for cytokine receptor signal transduction [41,42]. After activation, JAK2 kinase can phosphorylate STAT3, which then translocates to the nucleus to enhance the levels of downstream genes, including anti-apoptotic genes that are implicated in human cancer development [43–45]. Blocking upstream activators of STAT3 or suppressing phosphorylation of STAT3 itself has been considered to be a potential anticancer strategy [46–49]. A number of natural products have been reported to inhibit STAT3 activity and induce apoptosis in different tumor cell lines [10,50,51]. However, to our knowedge, few natural products act via JAK2 inhibition or have been investigated for the potential treatment of malignant melanoma [10,52]. WP1066, a small-molecule, directly process of tumorgenesis and metastasis by inhibiting the phosphorylation of JAK2/STAT3 pathway and the subsequent downstream protein, such as survivin and c-Myc, which are associated with tumor formation [53]. BBMD3, another JAK2 inhibitor, which inhibited autophosphorylation of JAK2 and blocked activation of downstream STAT3 signaling in melanoma cells [10]. In our previous research, we identified amentoflavone and its analogues as inhibitors of JAK2 activity with antiproliferative activity against the leukemic HEL cell line [34]. Due to the prevalance and high mortality rate of metatsatic melanoma, we were interested to investigate the efficacy of amentoflavone and its analogues against this type of cancer. In this study, we found that the amentoflavone analogue 1 showed more potent antiproliferative activity (0.36 μM) against melanoma cells compared to other cancer cell lines such as PC3, DU145, A2058 and HepG2 cells (Fig 2A–2E). Moreover, compound 1 had no discernible toxicity on normal LO2 cells (Fig 2F). This suggested that compound 1 could potentially be developed as a selective antitumor agent against malignant melanoma.
The mechanism of action of compound 1 was evaluated using multiple cellular assays. Compound 1 reduced the phosphorylation of JAK2 and STAT3 in A375 melanoma cells, but had no discernible effect on total JAK2 and STAT3 levels (Fig 3). The cellular thermal shift assay (CETSA) has been applied as the validation and optimization of drug target engagement [39]. An obvious shift of the melting temperature of JAK2 protein was detected (Fig 4), indicating that 1 could engage JAK2 even in the presence of cellular debris.
In addition, the phosphorylation of JAK2 and STAT3 can enhance the levels of the anti-apoptotic proteins Bcl-xl, Bcl-2, MCL-1 and survivin [54–56]. Therefore, we investigated whether compound 1 could induce apoptosis via acting through the JAK2/STAT3 pathway. Our results showed that compound 1 could induce apoptosis in approximately 80% of A375 cells after 24 h. Compound 1 could also induce cleavage of PARP and decrease caspase-3 levels, which could account for the pro-apoptotic activity of the compound (Fig 5). Subsequently, we studied whether compound 1 down-regulates the antiapoptotic protein Bcl-2 level which is regulated by STAT3 [54]. Our results showed that compound 1 inhibited the level of Bcl-2 in melanoma cells (Fig 6). This is consistent with other reports showing that inhibition of constitutively activated STAT3 correlates with altered Bcl-2/Bax levels [57]. HIF1α and ubiquitin are associated with apoptosis [58–60]. In order to rule out compound 1 acting via HIF1α or ubiquitin-associated pathways to induce apoptosis, the levels of HIF1α and ubiquitin were measured in treated cells. The results showed that 1 has no discernible effect on HIF1α and ubiquitin levels, suggesting that it did not induce apoptosis via those HIF1α and protein degradation pathways (Fig 7).
Taken together, these results clearly suggest that the apoptotic effect of compound 1 in melanoma cells is due to inhibition of JAK2/STAT3 signaling leading to the activation of pro-apopotic regulators and the inhibition of anti-apoptotic regulators, rather than through a general toxicity mechanism. To the best of our knowledge, this study is the first to demonstrate use of an amentoflavone analogue as a JAK2 inhibitor with pro-apoptotic and anti-proliferative effects against melanoma cells.
Acknowledgments
We especially thank Novartis Pharma AG for providing NVP-BBT594 for the biological assays.
Author Contributions
- Conceptualization: CHL DLM.
- Data curation: CHL DLM JJL XPC.
- Formal analysis: KJW JMH PC.
- Funding acquisition: CHL DLM.
- Investigation: QBH DWJK.
- Methodology: CHL DLM KJW JMH HJZ.
- Project administration: CHL DLM.
- Resources: CHL DLM PC.
- Software: KJW JMH ZZD.
- Supervision: CHL DLM.
- Validation: CHL DLM.
- Visualization: KJW JMH HJZ.
- Writing – original draft: KJW JMH.
- Writing – review & editing: HJZ KJW JMH KV.
References
- 1.
Situ BCI. Cancer Facts. 2015.
- 2. Gray-Schopfer V, Wellbrock C, Marais R. Melanoma biology and new targeted therapy. Nature. 2007; 445 (7130): 851–7. pmid:17314971
- 3. Lens M, Dawes M. Global perspectives of contemporary epidemiological trends of cutaneous malignant melanoma. British Journal of Dermatology. 2004; 150 (2): 179–85. pmid:14996086
- 4. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. New England Journal of Medicine. 2010; 363 (8): 711–23. pmid:20525992
- 5. Agarwala SS. Current systemic therapy for metastatic melanoma. Expert Review of Anticancer Therapy. 2009; 9 (5): 587–95. pmid:19445576
- 6. Eggermont AM, Kirkwood JM. Re-evaluating the role of dacarbazine in metastatic melanoma: what have we learned in 30 years? European Journal of Cancer. 2004; 40 (12): 1825–36. pmid:15288283
- 7. Petrella T, Quirt I, Verma S, Haynes AE, Charette M, Bak K, et al. Single-agent interleukin-2 in the treatment of metastatic melanoma: a systematic review. Cancer Treatment Reviews. 2007; 33 (5): 484–96. pmid:17562357
- 8. Trinh VA. Current management of metastatic melanoma. American Journal of Health-System Pharmacy. 2008; 65 (24): pS3–9.
- 9. De Nicolo A, Parisini E, Zhong Q, Dalla Palma M, Stoeckert KA, Domchek SM, et al. Multimodal assessment of protein functional deficiency supports pathogenicity of BRCA1 p. V1688del. Cancer Research. 2009; 69 (17): 7030–7. pmid:19706752
- 10. Nam S, Xie J, Perkins A, Ma Y, Yang F, Wu J, et al. Novel synthetic derivatives of the natural product berbamine inhibit Jak2/Stat3 signaling and induce apoptosis of human melanoma cells. Molecular Oncology. 2012; 6 (5): 484–93. pmid:22717603
- 11. Zhong Z, Wen Z, Darnell J. Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science. 1994; 264 (5155): 95–8. pmid:8140422
- 12. Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, et al. Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature. 1996; 379 (6566): 645. pmid:8628398
- 13. Andraos R, Qian Z, Bonenfant D, Rubert J, Vangrevelinghe E, Scheufler C, et al. Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent. Cancer Discovery. 2012; 2 (6): 512–23. pmid:22684457
- 14. O'Shea JJ, Schwartz DM, Villarino AV, Gadina M, McInnes IB, Laurence A. The JAK-STAT pathway: impact on human disease and therapeutic intervention. Annual Review of Medicine. 2015; 66: 311–28. pmid:25587654
- 15. Blaskovich MA, Sun J, Cantor A, Turkson J, Jove R, Sebti SM. Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice. Cancer Research. 2003; 63(6): 1270–9. pmid:12649187
- 16.
Leung P-C, Woo J, Kofler W. Annals of Traditional Chinese Medicine: Volume 6.
- 17. Aoki S, Yoshioka Y, Miyamoto Y, Higuchi K, Setiawan A, Murakami N, et al. Agosterol A, a novel polyhydroxylated sterol acetate reversing multidrug resistance from a marine sponge of Spongia sp. Tetrahedron Letters. 1998;39 (35): 6303–6.
- 18. Gomez C, Bai L, Zhang J, Nikolovska-Coleska Z, Chen J, Yi H, et al. Design, synthesis, and evaluation of peptidomimetics containing Freidinger lactams as STAT3 inhibitors. Bioorganic & Medicinal Chemistry Letters. 2009;19 (6): 1733–6.
- 19. Chen J, Bai L, Bernard D, Nikolovska-Coleska Z, Gomez C, Zhang J, et al. Structure-based design of conformationally constrained, cell-permeable STAT3 inhibitors. ACS Medicinal Chemistry Letters. 2010; 1(2): 85–9. pmid:20596242
- 20. Mosesso P, Bohm L, Pepe G, Fiore M, Carpinelli A, Gäde G, et al. Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro. Toxicology Letters. 2012; 213 (3): 361–6. pmid:22885097
- 21. Uzuner H, Bauer R, Fan T-P, Guo D-a, Dias A, El-Nezami H, et al. Traditional Chinese medicine research in the post-genomic era: good practice, priorities, challenges and opportunities. Journal of Ethnopharmacology. 2012; 140(3): 458–68. pmid:22387462
- 22. Yang C-Y, Wang S. Analysis of flexibility and hotspots in Bcl-xL and Mcl-1 proteins for the design of selective small-molecule inhibitors. ACS Medicinal Chemistry Letters. 2012; 3(4): 308–12. pmid:24900469
- 23. Yang D, Zhao Y, Li AY, Wang S, Wang G, Sun Y. Smac-mimetic compound SM-164 induces radiosensitization in breast cancer cells through activation of caspases and induction of apoptosis. Breast Cancer Research and Treatment. 2012; 133 (1): 189–99. pmid:21901386
- 24. Ye D, Shi Q, Leung C-H, Kim S-W, Park S-Y, Gullen EA, et al. Antitumor agents 294. Novel E-ring-modified camptothecin–4β-anilino-4′-O-demethyl-epipodophyllotoxin conjugates as DNA topoisomerase I inhibitors and cytotoxic agents. Bioorganic & Medicinal Chemistry. 2012; 20 (14): 4489–94.
- 25. Anreddy N, Gupta P, Kathawala RJ, Patel A, Wurpel JN, Chen Z-S. Tyrosine kinase inhibitors as reversal agents for ABC transporter mediated drug resistance. Molecules. 2014; 19 (9): 13848–77. pmid:25191874
- 26. Burslem GM, Kyle HF, Breeze AL, Edwards TA, Nelson A, Warriner SL, et al. Small‐Molecule Proteomimetic Inhibitors of the HIF‐1α–p300 Protein–Protein Interaction. ChemBioChem. 2014; 15 (8): 1083–7. pmid:24782431
- 27. Barnard A, Long K, Martin HL, Miles JA, Edwards TA, Tomlinson DC, et al. Selective and Potent Proteomimetic Inhibitors of Intracellular Protein–Protein Interactions. Angewandte Chemie International Edition. 2015; 54 (10): 2960–5. pmid:25651514
- 28. Hoffman-Luca CG, Ziazadeh D, McEachern D, Zhao Y, Sun W, Debussche L, et al. Elucidation of acquired resistance to Bcl-2 and MDM2 inhibitors in acute leukemia in vitro and in vivo. Clinical Cancer Research. 2015; 21 (11): 2558–68. pmid:25754349
- 29. Grison CM, Miles JA, Robin S, Wilson AJ, Aitken DJ. An α‐Helix‐Mimicking 12, 13‐Helix: Designed α/β/γ‐Foldamers as Selective Inhibitors of Protein–Protein Interactions. Angewandte Chemie International Edition. 2016; 128 (37): 11262–6.
- 30. Abraham I, Jain S, Wu C-P, Khanfar MA, Kuang Y, et al. Marine sponge-derived sipholane triterpenoids reverse P-glycoprotein (ABCB1)-mediated multidrug resistance in cancer cells. Biochemical Pharmacology. 2010; 80: 1497–1506. pmid:20696137
- 31. Sen R, Natarajan K, Bhullar J, Shukla S, Fang H-B, et al. The novel BCR-ABL and FLT3 inhibitor ponatinib is a potent inhibitor of the MDR-associated ATP-binding cassette transporter ABCG2. Molecular Cancer Therapeutics. 2012; 11: 2033–2044. pmid:22778153
- 32. Wang D-S, Patel A, Shukla S, Zhang Y-K, Wang Y-J, et al. Icotinib antagonizes ABCG2-mediated multidrug resistance, but not the pemetrexed resistance mediated by thymidylate synthase and ABCG2. Oncotarget. 2014; 5: 4529–4542. pmid:24980828
- 33. Zhang D-M, Shu C, Chen J-J, Sodani K, Wang J, et al. BBA, a derivative of 23-hydroxybetulinic acid, potently reverses ABCB1-mediated drug resistance in vitro and in vivo. Molecular Pharmaceutics. 2012; 9: 3147–3159. pmid:23046348
- 34. Ma D-L, Chan DS-H, Wei G, Zhong H-J, Yang H, et al. Virtual screening and optimization of Type II inhibitors of JAK2 from a natural product library. Chemical Communications. 2014; 50: 13885–13888. pmid:25225654
- 35. Zhong H-J, Lin S, Tam IL, Lu L, Chan DS-H, et al. In silico identification of natural product inhibitors of JAK2. Methods. 2015; 71: 21–25. pmid:25038528
- 36. Yang C, Wang W, Chen L, Liang J, Lin S, et al. Discovery of a VHL and HIF1α interaction inhibitor with in vivo angiogenic activity via structure-based virtual screening. Chemical Communications. 2016; 52: 12837–12840. pmid:27709157
- 37. Banerjee T, Van der Vliet A, Ziboh V. Downregulation of COX-2 and iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line. Prostaglandins, Leukotrienes and Essential Fatty Acids. 2002; 66 (5): 485–92.
- 38. Ren W, Qiao Z, Wang H, Zhu L, Zhang L. Flavonoids: promising anticancer agents. Medicinal Research Reviews. 2003; 23 (4): 519–34. pmid:12710022
- 39. Molina DM, Jafari R, Ignatushchenko M, Seki T, Larsson EA, et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science. 2013; 341: 84–87. pmid:23828940
- 40. Chen Q, Lu Z, Jin Y, Wu Y, Pan J. Triptolide inhibits Jak2 transcription and induces apoptosis in human myeloproliferative disorder cells bearing Jak2V617F through caspase-3-mediated cleavage of Mcl-1. Cancer Letters. 2010; 291 (2): 246–55. pmid:19942343
- 41. Rane SG, Reddy EP. Janus kinases: components of multiple signaling pathways. Oncogene. 2000;19(49):5662–79. pmid:11114747
- 42. Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nature Reviews Cancer. 2009; 9 (11): 798–809. pmid:19851315
- 43. Vainchenker W, Dusa A, Constantinescu SN, editors. JAKs in pathology: role of Janus kinases in hematopoietic malignancies and immunodeficiencies. Seminars in Cell & Developmental Biology. 2008; 19 (4): 389–93.
- 44. Kralovics R, Passamonti F, Buser AS, Teo S-S, Tiedt R, Passweg JR, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. New England Journal of Medicine. 2005; 352 (17): 1779–90. pmid:15858187
- 45. Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005; 7 (4): 387–97. pmid:15837627
- 46. Ahn KS, Sethi G, Sung B, Goel A, Ralhan R, Aggarwal BB. Guggulsterone, a farnesoid X receptor antagonist, inhibits constitutive and inducible STAT3 activation through induction of a protein tyrosine phosphatase SHP-1. Cancer Research. 2008; 68 (11): 4406–15. pmid:18519703
- 47. Bhutani M, Pathak AK, Nair AS, Kunnumakkara AB, Guha S, Sethi G, et al. Capsaicin is a novel blocker of constitutive and interleukin-6–inducible STAT3 activation. Clinical Cancer Research. 2007; 13 (10): 3024–32. pmid:17505005
- 48. Lin L, Amin R, Gallicano G, Glasgow E, Jogunoori W, Jessup J, et al. The STAT3 inhibitor NSC 74859 is effective in hepatocellular cancers with disrupted TGF-β signaling. Oncogene. 2009; 28 (7): 961–72. pmid:19137011
- 49. Kim M-J, Nam H-J, Kim H-P, Han S-W, Im S-A, Kim T-Y, et al. OPB-31121, a novel small molecular inhibitor, disrupts the JAK2/STAT3 pathway and exhibits an antitumor activity in gastric cancer cells. Cancer Letter. 2013; 335 (1): 145–52.
- 50. Tu SP, Jin H, Shi JD, Zhu LM, Suo Y, Lu G, et al. Curcumin induces the differentiation of myeloid-derived suppressor cells and inhibits their interaction with cancer cells and related tumor growth. Cancer Prevention Research. 2012; 5 (2): 205–15. pmid:22030090
- 51. Kim JE, Kim HS, Shin Y-J, Lee CS, Won C, Lee S-A, et al. LYR71, a derivative of trimeric resveratrol, inhibits tumorigenesis by blocking STAT3-mediated matrix metalloproteinase 9 expression. Experimental & Molecular Medicine. 2008; 40 (5): 514–22.
- 52. Boykin C, Zhang G, Chen Y, Zhang R, Fan X, Yang W, et al. Cucurbitacin IIa: a novel class of anti-cancer drug inducing non-reversible actin aggregation and inhibiting survivin independent of JAK2/STAT3 phosphorylation. British Journal of Cancer. 2011; 104 (5): 781–9. pmid:21304528
- 53. Kong L-Y, Abou-Ghazal MK, Wei J, Chakraborty A, Sun W, Qiao W, et al. A novel inhibitor of signal transducers and activators of transcription 3 activation is efficacious against established central nervous system melanoma and inhibits regulatory T cells. Clinical Cancer Research. 2008; 14 (18): 5759–68. pmid:18794085
- 54. Selvendiran K, Bratasz A, Tong L, Ignarro LJ, Kuppusamy P. NCX-4016, a nitro-derivative of aspirin, inhibits EGFR and STAT3 signaling and mdulates Bcl-2 proteins in cisplatin-resistant human ovarian cancer cells and xenografts. Cell Cycle. 2008; 7 (1): 81–8. pmid:18196976
- 55. Li F, Fernandez PP, Rajendran P, Hui KM, Sethi G. Diosgenin, a steroidal saponin, inhibits STAT3 signaling pathway leading to suppression of proliferation and chemosensitization of human hepatocellular carcinoma cells. Cancer Letters. 2010; 292 (2): 197–207. pmid:20053498
- 56. Dong Y, Lu B, Zhang X, Zhang J, Lai L, Li D, et al. Cucurbitacin E, a tetracyclic triterpenes compound from Chinese medicine, inhibits tumor angiogenesis through VEGFR2-mediated Jak2–STAT3 signaling pathway. Carcinogenesis. 2010; 31 (12):2097–104. pmid:20732905
- 57. Nielsen M, Kaestel C, Eriksen K, Woetmann A, Stokkedal T, Kaltoft K, et al. Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells. Leukemia. 1999; 13 (5): 735. pmid:10374878
- 58. Sutendra G, Dromparis P, Kinnaird A, Stenson T, Haromy A, et al. Mitochondrial activation by inhibition of PDKII suppresses HIF1a signaling and angiogenesis in cancer. Oncogene; 2013 32: 1638–1650. pmid:22614004
- 59. Swinson DE, Jones JL, Cox G, Richardson D, Harris AL, et al. Hypoxia‐inducible factor‐1α in non small cell lung cancer: Relation to growth factor, protease and apoptosis pathways. International Journal of Cancer; 2004 111: 43–50. pmid:15185341
- 60. Zhong H-J, Wang W, Kang T-S, Yan H, Yang Y, et al. A Rhodium (III) complex as an inhibitor of neural precursor cell expressed, developmentally down-regulated 8-activating enzyme with in vivo activity against inflammatory bowel disease. Journal of Medicinal Chemistry; 2016 60: 497–503. pmid:27976900