Discovery of a novel BTK inhibitor S-016 and identification of a new strategy for the treatment of lymphomas including BTK inhibitor-resistant lymphomas.

Bruton's tyrosine kinase (BTK) has emerged as a therapeutic target for B-cell malignancies, which is substantiated by the efficacy of various irreversible or reversible BTK inhibitors. However, on-target BTK mutations facilitating evasion from BTK inhibition lead to resistance that limits the therapeutic efficacy of BTK inhibitors. In this study we employed structure-based drug design strategies based on established BTK inhibitors and yielded a series of BTK targeting compounds. Among them, compound S-016 bearing a unique tricyclic structure exhibited potent BTK kinase inhibitory activity with an IC50 value of 0.5 nM, comparable to a commercially available BTK inhibitor ibrutinib (IC50 = 0.4 nM). S-016, as a novel irreversible BTK inhibitor, displayed superior kinase selectivity compared to ibrutinib and significant therapeutic effects against B-cell lymphoma both in vitro and in vivo. Furthermore, we generated BTK inhibitor-resistant lymphoma cells harboring BTK C481F or A428D to explore strategies for overcoming resistance. Co-culture of these DLBCL cells with M0 macrophages led to the polarization of M0 macrophages toward the M2 phenotype, a process known to support tumor progression. Intriguingly, we demonstrated that SYHA1813, a compound targeting both VEGFR and CSF1R, effectively reshaped the tumor microenvironment (TME) and significantly overcame the acquired resistance to BTK inhibitors in both BTK-mutated and wild-type BTK DLBCL models by inhibiting angiogenesis and modulating macrophage polarization. Overall, this study not only promotes the development of new BTK inhibitors but also offers innovative treatment strategies for B-cell lymphomas, including those with BTK mutations.

Discovery and biological evaluation of N-(3-(7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-4-methyl-2-oxo-2H-pyrimido[4,5-d][1,3]oxazin-1(4H)-yl)phenyl)acrylamide as potent Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine kinase (BTK) is a key component of the B cell receptor (BCR) signaling pathway and plays a crucial role in B cell malignancies and autoimmune disorders; thus, it is an attractive target for the treatment of B cell related diseases. Here, we evaluated the BTK inhibitory activity of a series of pyrimido[4,5-d][1,3]oxazin-2-one derivatives. Combining this evaluation with structure-activity relationship (SAR) analysis, we found that compound 2 exhibited potent BTK kinase inhibitory activity, with an IC50 of 7 nM. This derivative markedly inhibited BTK activation in TMD8 B cell lymphoma cells and thus inhibited the in vitro growth of the cells. Further studies revealed that compound 2 dose dependently arrested TMD8 cells at G1 phase, accompanied by decreased levels of Rb, phosphorylated Rb, and cyclin D1. Moreover, following treatment with compound 2, TMD8 cells underwent apoptosis associated with PARP and caspase 3 cleavage. Interestingly, the results of the kinase activity assay on a small panel of 35 kinases showed that the kinase selectivity of compound 2 was superior to that of the first-generation inhibitor ibrutinib, suggesting that compound 2 could be a second-generation inhibitor of BTK. In conclusion, we identified a potent and highly selective BTK inhibitor worthy of further development.

C11, a novel fibroblast growth factor receptor 1 (FGFR1) inhibitor, suppresses breast cancer metastasis and angiogenesis.

The fibroblast growth factor receptors (FGFRs) are increasingly considered attractive targets for therapeutic cancer intervention due to their roles in tumor metastasis and angiogenesis. Here, we identified a new selective FGFR inhibitor, C11, and assessed its antitumor activities. C11 was a selective FGFR1 inhibitor with an IC50 of 19 nM among a panel of 20 tyrosine kinases. C11 inhibited cell proliferation in various tumors, particularly bladder cancer and breast cancer. C11 also inhibited breast cancer MDA-MB-231 cell migration and invasion via suppression of FGFR1 phosphorylation and its downstream signaling pathway. Suppression of matrix metalloproteinases 2/9 (MMP2/9) was associated with the anti-motility activity of C11. Furthermore, the anti-angiogenesis activity of C11 was verified in endothelial cells and chicken chorioallantoic membranes (CAMs). C11 inhibited the migration and tube formation of HMEC-1 endothelial cells and inhibited angiogenesis in a CAM assay. In sum, C11 is a novel selective FGFR1 inhibitor that exhibits potent activity against breast cancer metastasis and angiogenesis.

Identification of compound D2923 as a novel anti-tumor agent targeting CSF1R.

Colony-stimulating factor 1 receptor (CSF1R) plays a critical role in promoting tumor progression in various types of tumors. Here, we identified D2923 as a novel and selective inhibitor of CSF1R and explored its antitumor activity both in vitro and in vivo. D2923 potently inhibited CSF1R in vitro kinase activity with an IC50 value of 0.3 nM. It exhibited 10- to 300-fold less potency against a panel of kinases tested. D2923 markedly blocked CSF-1-induced activation of CSF1R and its downstream signaling transduction in THP-1 and RAW264.7 macrophages and thus inhibited the in vitro growth of macrophages. Moreover, D2923 dose-dependently attenuated the proliferation of a small panel of myeloid leukemia cells, mainly by arresting the cells at G1 phase as well as inducing apoptosis in the cells. The results of the in vivo experiments further demonstrated that D2923 displayed potent antitumor activity against M-NFS-60 xenografts, with tumor growth inhibition rates of 50% and 88% at doses of 40 and 80 mg/kg, respectively. Additionally, D2923 was well tolerated with no significant body-weight loss observed in the treatment groups compared with the control. Furthermore, a western blot analysis and the immunohistochemistry results confirmed that the phosphorylation of CSF1R in tumor tissue was dramatically reduced after D2923 treatment, and this was accompanied by the depletion of macrophages in the tumor. Meanwhile, the expression of the proliferation marker Ki67 was also markedly decreased in the D2923 treatment group compared with the control group. Taken together, we identified D2923 as a novel and effective CSF1R inhibitor, which deserves further investigation.

7α,8α-Epoxynagilactones and their glucosides from the twigs of Podocarpus nagi: Isolation, structures, and cytotoxic activities.

A phytochemical investigation of twigs of Podocarpus nagi resulted in the identification of eight new type B nagilactones (1-8), all bearing a 7α,8α-epoxy-9(11)-enolide substructure, along with two known analogs (9-10). Their structures were determined on the basis of spectroscopic analysis, including HRESIMS, IR and NMR experiments, and X-ray crystallographic analysis. In vitro cytotoxic assay exhibited that compounds 1, 2, 9 and 10 could induce antiproliferation against three different types of human cancer cells while compounds 3 and 5 were inactive. Notably, the IC50 value of compound 1 is 0.208µM for A431 human epidermoid carcinoma cells, reaching the same level as the positive control combretastatin A-4 (0.104µM). Furthermore, compound 1 performed a strong inhibition of cancer cells by triggering apoptosis and arresting the cell cycle at G1 phase. These results unfold potential anticancer therapeutic applications of type B nagilactones.

New microtubulin inhibitor MT189 suppresses angiogenesis via the JNK-VEGF/VEGFR2 signaling axis.

The microtubulin inhibitor MT189 possesses anticancer activity and has been shown to overcome multidrug resistance. Here, we report that MT189 also inhibits angiogenesis. MT189 inhibited the proliferation, migration and differentiation of endothelial cells, with or without VEGF stimulation, and suppressed microvessel formation ex vivo and in vivo. MT189 reduced VEGF expression and secretion in both tumor and endothelial cells, under either hypoxic or normoxic conditions. The activation of VEGFR2 and downstream Src was thus abrogated in the MT189-treated endothelial cells. MT189 subsequently stabilized endothelial cell-cell junctions consist of VE-cadherin, ß-catenin, vinculin, and actin. MT189 also disrupted endothelial cell-matrix junctions by inhibiting the turnover of focal adhesions containing FAK, paxillin, vinculin, and actin. Inhibition of JNK reversed MT189-mediated inhibition of endothelial migration and differentiation, JNK activation, the reduction of VEGF expression and secretion, and the decrease of Src and FAK phosphorylation. These results indicate that MT189 suppresses angiogenesis by reducing endothelial proliferation, migration, and differentiation via the JNK-VEGF/VEGFR2 signaling axis. Together with our previous report showing that MT189 exhibited anticancer activity via the JNK-MCL-1 pathway, these new findings further support MT189-based drug development for cancer therapy.

Novel PARP1/2 inhibitor mefuparib hydrochloride elicits potent in vitro and in vivo anticancer activity, characteristic of high tissue distribution.

The approval of poly(ADP-ribose) polymerase (PARP) inhibitor AZD2281 in 2014 marked the successful establishment of the therapeutic strategy targeting homologous recombination repair defects of cancers in the clinic. However, AZD2281 has poor water solubility, low tissue distribution and relatively weak in vivo anticancer activity, which appears to become limiting factors for its clinical use. In this study, we found that mefuparib hydrochloride (MPH) was a potent PARP inhibitor, possessing prominent in vitro and in vivo anticancer activity. Notably, MPH displayed high water solubility (> 35 mg/ml) and potent PARP1/2 inhibition in a substrate-competitive manner. It reduced poly(ADP-ribose) (PAR) formation, enhanced γH2AX levels, induced G2/M arrest and subsequent apoptosis in homologous recombination repair (HR)-deficient cells. Proof-of-concept studies confirmed the MPH-caused synthetic lethality. MPH showed potent in vitro and in vivo proliferation and growth inhibition against HR-deficient cancer cells and synergistic sensitization of HR-proficient xenografts to the anticancer drug temozolomide. A good relationship between the anticancer activity and the PARP inhibition of MPH suggested that PAR formation and γH2AX accumulation could serve as its pharmacodynamic biomarkers. Its high bioavailability (40%~100%) and high tissue distribution in both monkeys and rats were its most important pharmacokinetic features. Its average concentrations were 33-fold higher in the tissues than in the plasma in rats. Our work supports the further clinical development of MPH as a novel PARP1/2 inhibitor for cancer therapy.

DW10075, a novel selective and small-molecule inhibitor of VEGFR, exhibits antitumor activities both in vitro and in vivo.

AIM: Targeting the VEGF/VEGF receptor (VEGFR) pathway has proved to be an effective antiangiogenic approach for cancer treatment. Here, we identified 6-((2-((3-acetamidophenyl)amino)pyrimidin-4-yl)oxy)-N-phenyl-1-naphthamide (designated herein as DW10075) as a novel and highly selective inhibitor of VEGFRs. METHODS: In vitro tyrosine kinase activity was measured using ELISA, and intracellular signaling pathway proteins were detected by Western blot analysis. Endothelial cell proliferation was examined with CCK-8 assays, and tumor cell proliferation was determined with SRB assays. Cell migration, tube formation and rat aortic ring assays were used to detect antiangiogenic activity. Antitumor efficacy was further evaluated in U87-MG human glioblastoma xenograft tumors in nude mice receiving DW10075 (500 mg · kg(-1) · d(-1), po) for two weeks. RESULTS: Among a panel of 21 kinases tested, DW10075 selectively inhibited VEGFR-1, VEGFR-2 and VEGFR-3 (the IC50 values were 6.4, 0.69 and 5.5 nmol/L, respectively), but did not affect 18 other kinases including FGFR and PDGFR at 10 µmol/L. DW10075 significantly blocked VEGF-induced activation of VEGFR and its downstream signaling transduction in primary human umbilical vein endothelial cells (HUVECs), thus inhibited VEGF-induced HUVEC proliferation. DW10075 (1-100 nmol/L) dose-dependently inhibited VEGF-induced HUVEC migration and tube formation and suppressed angiogenesis in both the rat aortic ring model and the chicken chorioallantoic membrane model. Furthermore, DW10075 exhibited anti-proliferative activity against 22 different human cancer cell lines with IC50 values ranging from 2.2 µmol/L (for U87-MG human glioblastoma cells) to 22.2 µmol/L (for A375 melanoma cells). In U87-MG xenograft tumors in nude mice, oral administration of DW10075 significantly suppressed tumor growth, and reduced the expression of CD31 and Ki67 in the tumor tissues. CONCLUSION: DW10075 is a potent and highly selective inhibitor of VEGFR that deserves further development.

DCLAK11, a multi-tyrosine kinase inhibitor, exhibits potent antitumor and antiangiogenic activity in vitro.

AIM: To investigate the molecular targets of DCLAK11, a novel compound discovered from a series of substituted pyridin-3-amine derivatives, and to characterize its anti-tumor properties in vitro. METHODS: Kinase inhibition was measured by an ELISA assay. Cell viability was assessed with an SRB or a CCK8 assay. The alterations induced by kinase signaling proteins in cancer cells were detected by Western blot. Apoptosis was determined by an Annexin V-PI assay. The following assays were used to evaluate the impact on angiogenesis: wound-healing, Transwell, tube formation and microvessel outgrowth from rat aortic rings. RESULTS: DCLAK11 was a multi-targeted kinase inhibitor that primarily inhibited the EGFR, HER2, and VEGFR2 tyrosine kinases with IC50 value of 6.5, 18, and 31 nmol/L, respectively. DCLAK11 potently inhibited the proliferation of EGFR- and HER2-driven cancer cells: its IC50 value was 12 and 22 nmol/L, respectively, in HCC827 and HCC4006 cells with EGFR exon deletions, and 19 and 81 nmol/L, respectively, in NCI-N87 and BT474 cells with HER2 amplification. Consistently, DCLAK11 blocked the EGFR and HER2 signaling in cancer cells with either an EGFR or a HER2 aberration. Furthermore, DCLAK11 effectively induced EGFR/HER2-driven cell apoptosis. Moreover, DCLAK11 exhibited anti-angiogenic activity, as shown by its inhibitory effect on the proliferation, migration and tube formation of human umbilical vascular endothelial cells and the microvessel outgrowth of rat aortic rings. CONCLUSIONS: DCLAK11 is a multi-targeted kinase inhibitor with remarkable potency against tyrosine kinases EGFR, HER2 and VEGFR2, which confirms its potent anti-cancer activity in EGFR- and HER2-addicted cancers and its anti-angiogenic activity.

Topoisomerase II inhibitors from the roots of Stellera chamaejasme L.

Three new compounds, including one daphnane diterpene (1), one sesquiterpene (6), and one lignan (7) have been isolated from the Stellera chamaejasme L., together with five other known compounds, including four daphnane diterpenenoids (2-5) and one lignan (8). The structures of the new compounds were elucidated by spectroscopic analysis. The cytotoxicities of compounds 1-8 towards human lung adenocarcinoma cells (A549 cells) were evaluated using a sulforhodamine B assay. All of the compounds displayed significant cytotoxicity, with IC50 values in the ranging of 0.2 nM to 2.0 µM. Mechanistic studies revealed that the antitumor activities of compounds 1-3 and 7 were derived from their inhibition of topoisomerase II (Topo II). Furthermore, as a Topo II inhibitor, compound 1 was found to effectively induced G2-M phase cell cycle arrest and apoptosis in cancer cells.

Identification of DW532 as a novel anti-tumor agent targeting both kinases and tubulin.

AIM: 7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-2H-chromen-2-one (DW532) is one of simplified analogues of hematoxylin that has shown broad-spectrum inhibition on tyrosine kinases and in vitro anti-cancer activities. The aim of this study was to identify DW532 as a agent targeting both kinases and tubulin, and to investigate its anti-cancer and anti-angiogenesis activities. METHODS: In vitro tyrosine kinases activity was examined with ELISA, and tyrosine kinases activity in cells was evaluated with Western blot analysis. Tubulin turbidity assay, surface plasmon resonance and immunofluorescence technique were used to characterize the tubulin inhibitory activity. Cell proliferation was examined with SRB assay, and cell apoptosis and cell cycle distribution were analyzed with Annexin-V/PI staining and flow cytometry. Tube formation, aortic ring and chick chorioallantoic membrane assays were used to evaluate the anti-angiogenesis efficacy. RESULTS: DW532 inhibited EGFR and VEGFR2 in vitro kinase activity (the IC50 values were 4.9 and 5.5 µmol/L, respectively), and suppressed their downstream signaling. DW532 dose-dependently inhibited tubulin polymerization via direct binding to tubulin, thus disrupting the mitotic spindle assembly and leading to abnormal cell division. In a panel of human cancer cells, DW532 (1 and 10 µmol/L) induced G2/M phase arrest and cell apoptosis, which subsequently resulted in cytotoxicity. Knockdown of BubR1 or Mps1, the two core proteins of the spindle assembly checkpoint dramatically decreased DW532-induced cell cycle arrest in MDA-MB-468 cells. Moreover, treatment with DW532 potently and dose-dependently suppressed angiogenesis in vitro and in vivo. CONCLUSION: DW532 is a dual inhibitor against tubulin and tyrosine kinases, and deserves further development as a novel anti-cancer agent.

MCL-1 degradation mediated by JNK activation via MEKK1/TAK1-MKK4 contributes to anticancer activity of new tubulin inhibitor MT189.

Colchicine site-targeted tubulin inhibitors are a promising type of anticancer drugs. MT189 is a new derivative of MT119, a previously reported colchicine site-binding antitubulin agent. In this study, MT189 was demonstrated to retain the property of MT119 in disrupting microtubulin via binding to the colchicine site, causing mitotic arrest and inducing apoptosis, and to display 8.7-fold enhanced proliferative inhibition in a panel of cancer cells. MT189 was shown to elicit in vivo anticancer effects on MDA-MB-231 xenografts in nude mice, and the tumor growth was suppressed by 35.9% over 14 days. MT189 led to degradation of MCL-1, a member of the antiapoptotic BCL-2 protein family. Its overexpression reduced but its silenced expression increased the apoptotic induction followed by the treatment with MT189. Moreover, the treatment with MT189 caused activation of the MEKK1/TAK1-MKK4-JNK signaling pathway. The activated JNK resulted in phosphorylation of MCL-1, which facilitated its ubiquitination-mediated degradation. Our results show that MT189 inhibits microtubulin polymerization by binding to the colchicine site. Relief of apoptotic suppression by MCL-1 degradation together with mitotic arrest contributes to the anticancer activity of MT189.

Systematic combination screening reveals synergism between rapamycin and sunitinib against human lung cancer.

Mammalian target of rapamycin (mTOR) acts as a hub integrating signals from nutrient availability and growth factors and plays central roles in regulating protein synthesis and cell growth, which has been validated as a promising target for cancer therapy. Rapamycin and its analogues have emerged as the first generation of mTOR inhibitors, but their efficacy is modest in clinical settings. Combinatorial use of rapamycin with other drugs is a promising strategy to improve its anticancer activity. Here we developed an unbiased systematic binary screening platform aiming to discover new remedy for rapamycin-based cancer therapy. We found that sunitinib emerged as one of the clinically available anticancer drugs screened that displayed significant synergy with rapamycin in NSCLC cells. Combination of rapamycin with sunitinib resulted in enhanced cell cycle arrest in G1 phase, which was accompanied with enhanced suppression of mTOR signaling and disruption of the negative feedback loop that activate AKT upon mTORC1 inhibition. Furthermore, sunitinib and rapamycin displayed synergistic activity against tube formation by human microvessel endothelial cells as well as outgrowth of endothelial tubes and microvessels both in vitro and in vivo, which is associated with down-regulation of VEGF secretion and HIF1α expression. Our study demonstrated that new combinatorial regimen could be identified via systematic drug combination screening and established a mechanistic rationale for a combination approach using rapalogs and sunitinib in the treatment of human NSCLC.

DW09849, a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, prevents PI3K signaling and preferentially inhibits proliferation of cells containing the oncogenic mutation p110α (H1047R).

Phosphatidylinositol 3-kinase, α isoform (PI3Kα) plays essential roles in cell metabolism, growth, and proliferation and has been validated as a promising anticancer target. In an effort to search for new PI3Kα-selective inhibitors, DW series compounds were designed and synthesized aiming to reduce the off-target effects of their parent compound PIK-75 [2-methyl-5-nitro-1-benzenesulfonic acid 2-[(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene]-1-methylhydrazide], which was reported to selectively target PI3Kα. A series of compounds named DW series potently inhibited the kinase activity of PI3Kα with little activity against PI3K-related protein kinases and a panel of 15 tyrosine kinases. Similar to PIK-75, DW series compounds were more potent to inhibit PI3Kα among four class I PI3K isoforms, whereas a representative compound DW09849 [(E)-N'-((6-bromoimidazo[1,2-a]pyridin-3-yl)methylene)-N-ethyl-2-methyl-5-nitrobenzohydrazide] displayed distinct binding mode compared with PIK-75. Although DW series compounds inhibited proliferation of rhabdomyosarcoma RH30 cells at elevated 50% inhibitory concentrations (IC50) in comparison with PIK-75, they were more selective than PIK-75 to inhibit PI3K signaling in the cellular context. In particular, DW09849 significantly and persistently blocked PI3K/protein kinase B signaling in RH30 cells, which consequently arrested RH30 cells in the G1 phase. Moreover, DW09849 selectively suppressed the proliferation and clonogenesis of transformed RK3E/HR cells harboring oncogenic mutation of p110α H1047R, as well as a panel of human breast cancer cells containing mutated PI3Kα, which is consistent with the finding that DW09849 demonstrated preference against H1047R mutated PI3Kα in molecular docking stimulation. These results suggest that DW series compounds, especially DW09849, selectively targeting PI3Kα with less off-target effects than PIK-75, provide new clues for the design and discovery of new specific PI3Kα inhibitors for cancer therapy.

Characterization of the conversion between CD133+ and CD133- cells in colon cancer SW620 cell line.

The state of cancer stem cells (CSC) under reversible fluctuations, which has been revealed in breast cancer cells most recently, suggests that subpopulations with distinct phenotypes and functions within cancer cells can undergo inter-conversion. To investigate the possibility in colon cancer cells, we employed CD133 as the CSC marker, and characterized CD133 expression pattern and the biological features of the CD133 (+) and CD133 (-) subsets. Flow cytometry revealed that CD133 was bimodally expressed in SW620 cells among eight colon cancer cell lines. The CD133 (+) clonal SW620 cells displayed a differential gene expression profile, higher cellular reactive oxygen species (ROS), enhanced tumorigenesis and resistance to 5-fluorouracil. The conversion in term of the CD133 phenotype of the sorted cells was observed in vitro and in vivo. The fraction of the CD133 (+) cells decreased from 99% to 80% in the sorted CD133 (+) population while rising from 5 to 10% in the sorted CD133 (-) population during the first 20-day cultivation and then stayed almost unchanged. A fraction (about 20%) of the CD133 (+) clonal cells lost their CD133 marker while about 10% of the CD133 (-) clonal cells acquired the CD133 marker. 5-Azacytidine enhanced the fraction of the CD133 (+) cells in both of the CD133 (+) and CD133 (-) clonal cells. Our data demonstrate that CD133 expression is dynamic and reversible, and reveal the inter-conversion between the CD133 (+) and the CD133 (-) SW620 cells, suggesting that the CD133 phenotype of SW620 cell population is retained by the conversion between the two cell subsets.

Establishment of platform for screening insulin-like growth factor-1 receptor inhibitors and evaluation of novel inhibitors.

AIM: The insulin-like growth factor-1 receptor (IGF1R) is over-expressed in a wide variety of tumors and contributes to tumor cell proliferation, metastasis and drug resistance. The aim of this study was to establish a sensitive screening platform to identify novel IGF1R inhibitors. METHODS: The catalytic domain of IGF1R was expressed using the Bac-to-Bac baculovirus expression system. The screening platform for IGF1R inhibitors was established based on ELISA. The binding profile of IGF1R with the inhibitors was predicted with molecular docking and then subjected to the surface plasmon resonance (SPR) approach. The growth inhibition of cancer cells by the inhibitors was assessed with MTT assay. Apoptosis was analyzed using flow cytometry and Western blotting. RESULTS: A naturally occurring small molecule compound hematoxylin was identified as the most potent inhibitor (IC50 value=1.8±0.1 µmol/L) within a library of more than 200 compounds tested. Molecular simulation predicted the possible binding mode of hematoxylin with IGF1R. An SPR assay further confirmed that hematoxylin bound directly to IGF1R with high binding affinity (Kd=4.2 × 10⁻6 mol/L). In HL-60 cancer cells, hematoxylin inactivated the phosphorylation of IGF1R and downstream signaling and therefore suppressed cell proliferation. Mechanistic studies revealed that hematoxylin induced apoptosis in HL-60 cells via both extrinsic and intrinsic pathways. CONCLUSION: A simple, sensitive ELISA-based screening platform for identifying IGF1R inhibitors was established. Hematoxylin was identified as a promising IGF1R inhibitor with effective antitumor activity that deserves further investigation.

Drug transporter-independent liver cancer cell killing by a marine steroid methyl spongoate via apoptosis induction.

Hepatocellular carcinoma (HCC) is inherently resistant to the majority of clinical anticancer drugs. To obtain drugs that can circumvent or evade such inherent drug resistance of HCC, we investigated the effect of the marinely derived steroid methyl spongoate (MESP) on HCC cells. MESP displayed potent cell killing against a panel of six HCC cell lines, independent of their expression of drug transporters. MESP did not change the function of the drug transporters, and its cell killing was not impaired in multidrug-resistant cancer cells overexpressing the transporters. The cell killing of MESP was irrelevant to estrogen or androgen signaling and was not associated with cell cycle progression, inhibition of microtubules, and topoisomerases. In contrast, MESP potently induced apoptosis via activation of a proapoptotic caspase cascade and relief of the suppression of antiapoptotic signal transducers and activators of transcription 3 (STAT3) signaling. MESP inhibited the phosphorylation of STAT3, a critical survival signaling factor that reduced the expression of the antiapoptotic protein x-linked inhibitor of apoptosis protein but enhanced the expression of the proapoptotic protein Bax, thus promoting caspase-dependent apoptosis. These data reveal that MESP may well serve as an important candidate drug lead for HCC therapy.

Increased accumulation of hypoxia-inducible factor-1α with reduced transcriptional activity mediates the antitumor effect of triptolide.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α), a critical transcription factor to reduced O2 availability, has been demonstrated to be extensively involved in tumor survival, aggressive progression, drug resistance and angiogenesis. Thus it has been considered as a potential anticancer target. Triptolide is the main principle responsible for the biological activities of the Traditional Chinese Medicine tripterygium wilfordii Hook F. Triptolide possesses great chemotherapy potential for cancer with its broad-spectrum anticancer, antiangiogenesis, and drug-resistance circumvention activities. Numerous biological molecules inhibited by triptolide have been viewed as its possible targets. However, the anticancer action mechanisms of triptolide remains to be further investigated. Here we used human ovarian SKOV-3 cancer cells as a model to probe the effect of triptolide on HIF-1α. RESULTS: Triptolide was observed to inhibit the proliferation of SKOV-3 cells, and meanwhile, to enhance the accumulation of HIF-1α protein in SKOV-3, A549 and DU145 cells under different conditions. Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells. However, triptolide was found to increase the levels of HIF-1α mRNA. Unexpectedly, the HIF-1α protein induced by triptolide appeared to lose its transcriptional activity, as evidenced by the decreased mRNA levels of its target genes including VEGF, BNIP3 and CAIX. The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene. Moreover, the silencing of HIF-1α partially prevented the cytotoxicity and apoptosis triggered by triptolide. CONCLUSIONS: The potent induction of HIF-1α protein involved in its cytotoxicity, together with the suppression of HIF-1 transcriptional activity, indicates the great therapeutic potential of triptolide as an anticancer drug. Meanwhile, our data further stress the possibility that HIF-1α functions in an unresolved nature or condition.

WJD008, a dual phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin inhibitor, prevents PI3K signaling and inhibits the proliferation of transformed cells with oncogenic PI3K mutant.

The phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway is often constitutively activated in various human cancers, providing validated targets for cancer therapy. Among a series of 5-cyano-6-morpholino-4-substituted-pyrimidine analogs designed and synthesized based on PI3K target, 4-(2-(dimethylamino)vinyl)-2-(3-hydroxyphenyl)-6-morpholinopyrimidine-5-carbonitrile (WJD008) was selected for further pharmacological characterization because of its potent activity against PI3K signaling. WJD008 inhibited kinase activity of PI3Kalpha and mTOR with less activity against PIKK family members. In cellular settings, WJD008 abrogated insulin-like growth factor-I-activated PI3K-Akt-mTOR signaling cascade and blocked the membrane translocation of a pleckstrin homology domain containing enhanced green fluorescent protein-general receptor for phosphoinositides, isoform 1-pleckstrin homology fusion protein, suggesting down-regulation of phosphatidylinositol (3,4,5)-trisphosphate output induced by WJD008 resulted in inactivation of PI3K pathway. Consequently, WJD008 arrested cells in G(1) phase without induction of apoptosis. Furthermore, WJD008 reversed the hyperactivation of the PI3K pathway caused by the oncogenic mutation of p110alpha H1047R and suppressed the proliferation and clonogenesis of transformed RK3E cells harboring this mutant. WJD008 was superior to the pan-PI3K inhibitor wortmannin against proliferation of a panel of cancer cells independently of their status of PI3K pathway or tissue originations. In summary, WJD008 is a potent dual PI3K/mTOR modulator with antiproliferative and anticlonogenic activity in tumor cells and transformed cells with PIK3CA mutant, which provides new clues for the design and development of this chemical scaffold as an anticancer drug.