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T–lymphokine-activated killer cell–originated protein kinase (TOPK) is a serine-threonine mitogen-activated protein kinase that is highly expressed in many types of human cancer. Due to its important role in cancer progression, TOPK is becoming an attractive target in chemotherapeutic drug design. In this study, a series of 1-phenyl phenanthridin-6(5H)-one derivatives have been identified as a novel chemical class of TOPK inhibitors. Some of them displayed very potent anti-cancer activity with IC50s less than 100 nM, superior than reference compound OTS964. The most potent compound, 9g suppressed the growth of cancer cells by apoptosis and specifically inhibited the activities of TOPK. Oral administration of 9g effectively suppressed tumor growth with TGI >79.7% in colorectal cancer xenograft models, demonstrating superior efficacy compared to OTS964. Pharmacokinetic studies reveal its good oral bioavailability. Our findings therefore show that 9g is a specific inhibitor of TOPK both in vitro and in vivo that may be further developed as a potential therapeutic agent against colorectal cancer.

Several FDA approved small molecule anti-cancer drugs contain indazole scaffolds. Here, we report the design, synthesis and biological evaluation of a series of indazole derivatives. In vitro antiproliferative activity screening showed that compound 2f had potent growth inhibitory activity against several cancer cell lines (IC50 = 0.23–1.15 μM). Treatment of the breast cancer cell line 4T1 with 2f inhibited cell proliferation and colony formation. 2f dose-dependently promoted the apoptosis of 4T1 cells, which was connected with the upregulation of cleaved caspase-3 and Bax, and downregulation of Bcl-2. 2f also decreased the mitochondrial membrane potential and increased the levels of reactive oxygen species (ROS) in 4T1 cells. Additionally, treatment with 2f disrupted 4T1 cells migration and invasion, and the reduction of matrix metalloproteinase metalloproteinase-9 (MMP9) and increase of tissue inhibitor matrix metalloproteinase 2 (TIMP2) were also observed. Moreover, 2f could suppress the growth of the 4T1 tumor model without obvious side effects in vivo. Taken together, these results identified 2f as a potential small molecule anti-cancer agent.

Aberrant signaling of fibroblast growth factor receptors (FGFRs) has been identified as a driver of tumorigenesis and the development of many solid tumors, making FGFRs a compelling target for anticancer therapy. Herein, we describe the design and synthesis of pyrido[1,2-a]pyrimidinone derivatives as potent FGFR inhibitors. Examination of structure–activity relationships and preliminary assessment identified 23d as a novel FGFR inhibitor that displayed excellent potency in vitro. Candidate 23d suppressed the phosphorylation of FGFR signaling pathways and induced cell cycle arrest and apoptosis at low nanomolar concentration. In the kinase inhibition profile, 23d showed excellent kinase selectivity for the FGFR family. Furthermore, 23d showed higher aqueous solubility than Erdafitinib. Moreover, 23d exhibited potent antitumor activity (tumor growth inhibition = 106.4%) in FGFR2-amplified SNU-16 gastric cancer xenograft model using a daily oral dose of 30 mg/kg. These results suggest that 23d is a promising candidate for further drug development.

A novel series of 4-(4-formamidophenylamino)-N-methylpicolinamide derivatives were synthesized and evaluated against different tumor cell lines. Experiments in vitro showed that these derivatives could inhibit the proliferation of two kinds of human cancer cell lines (HepG2, HCT116) at low micromolar concentrations and the most potent analog 5q possessed broad-spectrum antiproliferative activity. Experiments in vivo demonstrated that 5q could effectively prolong the longevity of colon carcinoma-burdened mice and slow down the progression of cancer cells by suppression of angiogenesis and the induction of apoptosis and necrosis.

Polo-like kinase 4 (PLK4), a member of the polo-like kinase family, plays several important roles in mitotic regulation, including centrosome duplication, spindle formation, and cytokinesis. PLK4 overexpression is frequently detected in many human cancers, including ovarian cancer, and the inhibition of PLK4 activity results in cancer cell mitotic arrest and apoptosis. Therefore, PLK4 might be a valid therapeutic target for antitumor therapy. In the present study, we aimed to determine if YLZ-F5, a potent small-molecule inhibitor of PLK4, inhibits ovarian cancer cell growth. MTT assay showed that YLZ-F5 inhibited ovarian cancer cell proliferation in a concentration- and time-dependent manner. The results of colony formation assays were consistent with those of the MTT assay results. In addition, YLZ-F5 induced ovarian cancer cell apoptosis that was associated with activation of caspase-3/caspase-9. Moreover, YLZ-F5 caused aberrant in centriole duplication that was associated with the inhibition of PLK4 phosphorylation. Notably, we showed that YLZ-F5 promoted the accumulation of ovarian cancer cells with mitotic defects (> 4 N DNA content) in a concentration-dependent manner. Furthermore, YLZ-F5 markedly inhibited the migration of A2780 cells. Taken together, these findings suggest that YLZ-F5 is a potential drug candidate for human ovarian cancer.

Polo-like kinase 4 (PLK4) is indispensable for precise control of centriole duplication. Abnormal expression of PLK4 has been reported in many human cancers, and inhibition of PLK4 activity results in their mitotic arrest and apoptosis. Therefore, PLK4 may be a valid therapeutic target for antitumor therapy. However, clinically available small-molecule inhibitors targeting PLK4 are deficient and their underlying mechanisms still remain not fully clear. Herein, the effects of YLT-11 on breast cancer cells and the associated mechanism were investigated. In vitro, YLT-11 exhibited significant antiproliferation activities against breast cancer cells. Meanwhile, cells treated with YLT-11 exhibited effects consistent with PLK4 kinase inhibition, including dysregulated centriole duplication and mitotic defects, sequentially making tumor cells more vulnerable to chemotherapy. Furthermore, YLT-11 could strongly regulate downstream factors of PLK4, which was involved in cell cycle regulation, ultimately inducing apoptosis of breast cancer cell. In vivo, oral administration of YLT-11 significantly suppressed the tumor growth in human breast cancer xenograft models at doses that are well tolerated. In summary, the preclinical data show that YLT-11 could be a promising candidate drug for breast tumor therapy.

Polo-like kinase 4 (PLK4), a vital regulator of centriole duplication, is important for maintaining genome stability. Dysregulation of PLK4 has been found in several human cancers and is associated with a predisposition to tumorigenesis. Herein, we describe the discovery of (E)-4-(3-arylvinyl-1H-indazol-6-yl)pyrimidin-2-amine derivatives as potent PLK4 inhibitors with more concise structure using a scaffold hopping strategy. SAR exploration and preliminary assessment identified 14i as a new PLK4 inhibitor which displayed excellent potency in vitro. 14i could inhibit the activity of PLK4, perturb centriole replication, result in mitosis disorder and induce cell apoptosis in breast cancer cells. Moreover 14i demonstrated significant antitumor efficacy in the MDA-MB-468 and MDA-MB-231 xenograft models. This study suggested that this concise chemotype would represent a promising scaffold of PLK4 inhibitors for cancer therapy and 14i would be an attractive lead compound for further optimization and evaluation.

Enhancer of zeste homolog 2 (EZH2) serves as the catalytic subunit of the polycomb repression complex 2 (PRC2), which is implicated in cancer progression metastasis and poor prognosis. Based on our EZH2 inhibitor SKLB1049 with low nanomolar activity, we extended the “tail” region to get a series of (E)-1,2-diphenylethene derivatives as novel EZH2 inhibitors. SAR exploration and preliminary assessment led to the discovery of the potent novel EZH2 inhibitor 9b (EZH2WT IC50 = 22.0 nM). Compound 9b inhibited the proliferation of WSU-DLCL2 and SU-DHL-4 cell lines (IC50 = 1.61 µM and 2.34 µM, respectively). The biological evaluation showed that 9b was a potent inhibitor for wild-type EZH2 and greatly reduced the overall levels of H3K27me3 in a concentration-dependent manner. Further study indicated that 9b could significantly induce apoptosis of SU-DHL-4 cells. These findings indicated that 9b would be an attractive lead compound for further optimization and evaluation.

Castration-resistant prostate cancer (CRPC) is a lethal disease with few treatment alternatives once patients become resistant to second-generation antiandrogens. In CRPC, BET proteins are key regulators of AR- and MYC-mediated transcription, while the PLK1 inhibitor potentially downregulates AR and MYC besides influencing the cell cycle. Therefore, synchronous inhibition of BET and PLK1 would be a promising approach for CRPC therapy. This study developed a dual BET and PLK1 inhibitor WNY0824 with nanomolar and equipotent inhibition of BRD4 and PLK1. In vitro, WNY0824 exhibited excellent antiproliferation activity on AR-positive CRPC cells and induced apoptosis. These activities are attributable to its disruption of the AR-transcriptional program and the inhibition of the ETS pathway. Furthermore, WNY0824 downregulated MYC and induced mitotic abnormality. In vivo, oral WNY0824 administration suppressed tumor growth in the CRPC xenograft model of enzalutamide resistance. These findings suggest that WNY0824 is a selective dual BET and PLK1 inhibitor with potent anti-CRPC oncogenic activity and provides insights into the development of other novel dual BET- and PLK1-inhibiting drugs.

Colorectal carcinoma (CRC) is one of the most common cancers with high metastatic potential, explaining why identifying new drug candidates that inhibit tumour metastasis is an urgent need. The aim of this study was to evaluate the biological activities of pectolinarigenin (PEC, a natural flavonoid present in Cirsium chanroenicum) in CRC in vitro and in vivo and to determine its underlying mechanism of action. Here, we observed that treatment with PEC could inhibit cell viability and induce apoptosis in cancer cells in a concentration- and time-dependent manner. The occurrence of apoptosis was associated with activation of caspase-3 and Bax and decreased expression of Bcl-2. In addition, PEC markedly impaired CRC cell migration and invasion by downregulating the expression of matrix metalloproteinase (MMP-9) and phosphorylated-Stat3Tyr705. Moreover, our studies showed that PEC inhibited abdominal metastasis models of murine colorectal cancer. In addition, histological and immunohistochemical analyses revealed a decrease in Ki67-positive cells, MMP9-positive cells and p-Stat3Tyr705 cells upon treatment with PEC compared to control samples. Furthermore, PEC reduced the number of myeloid-derived suppressor cells (MDSCs) in the blood and tumours, which was accompanied by the increased infiltration of CD8+T cells in the blood. Taken together, our findings suggested that PEC could be used as a natural drug to inhibit CRC metastasis.

PI3Kδ has proved to be an effective target for anti-lymphoma drugs. However, the application of current approved PI3Kδ inhibitors has been greatly limited due to their specific immune-mediated toxicity and increased risk of infection, it is necessary to develop more PI3Kδ inhibitors with new scaffold. In this study, SAR study with respect to piperazinone-containing purine derivatives led to the discovery of a potent and selective PI3Kδ inhibitor, 4-(cyclobutanecarbonyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (WNY1613). WNY1613 exhibits good antiproliferative activity against a panel of non-Hodgkin's lymphoma (NHL) cell lines by inducing cancer cell apoptosis and inhibiting the phosphorylation of PI3K and MAPK downstream components. In addition, it can also prevent the tumor growth in both SU-DHL-6 and JEKO-1 xenograft models without observable toxicity. WNY1613 thus could be developed as a promising candidate for the treatment of NHL after subsequent extensive pharmacodynamics and pharmacokinetics investigation.

Background: Cancer is still a major public health issue worldwide, and new therapeutics with anti-tumor activity are still urgently needed. Methods: The anti-tumor activity of TH-39, which shows potent anti-proliferative activity against K562 cells with an IC50 of 0.78 µM, was investigated using immunoblot, co-immunoprecipitation, the MTT assay, and flow cytometry. Results: Mechanistically, TH-39 may disrupt the interaction between Hec1 and Nek2 in K562 cells. Moreover, TH-39 inhibited cell proliferation in a concentration- and time-dependent manner by influencing the morphology of K562 cells and inducing G0/G1 phase arrest. G0/G1 phase arrest was associated with down-regulation of CDK2-cyclin E complex and CDK4/6-cyclin D complex activities. Furthermore, TH-39 also induced cell apoptosis, which was associated with activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax. TH-39 could also decrease mitochondrial membrane potential (Δψm) and increase reactive oxygen species (ROS) accumulation in K562 cells. The results indicated that TH-39 might induce apoptosis via the ROS-mitochondrial apoptotic pathway. Conclusion: This study highlights the potential therapeutic efficacy of the anti-cancer compound TH-39 in treatment-resistant chronic myeloid leukemia.

Pancreatic carcinoma is a still unsolved health problem all over the world with poor prognosis and high mortality rate. YLT256, a novel synthesized chemical small inhibitor, displays potent antineoplastic activities via inducing apoptosis both in vitro and in vivo. In this study, we found that YLT256 showed growth inhibition against a broad spectrum of human cancer cell lines and pancreatic cancer cell line BxPc-3 was the most sensitive with an IC50 of 0.42μM. We also found YLT256 could induce apoptosis of BxPc-3 cells in a dose-dependent manner. Western blot analysis revealed that the occurrence of its apoptosis was associated with activation of caspases-3 and -9, up-regulation of pro-apoptotic Bak, and down-regulation of anti-apoptotic Bcl-2. Moreover, YLT256-treated resulted in changes of mitochondrial membrane potential (Δψm), and generation of reactive oxygen species (ROS). Furthermore, our data also revealed that YLT256 suppressed the growth of established tumor-bearing xenograft models without obvious side effects. Immunohistochemical analyses and TUNEL assay revealed an increase in cleaved caspase-3-positive cells and TUNEL-positive cells, a decrease in Ki67-positive cells upon YLT256. Together, all the results of present study provided evidence demonstrating that YLT256 could be a promising potential drug candidate for pancreatic cancer therapy.

A series of 3-(((9H-purin-6-yl) amino) methyl) pyridin-2(1H)-one derivatives were designed, synthesized and confirmed as tubulin polymerization inhibitors. All compounds were evaluated for their anti-proliferative activities on three colorectal carcinoma (CRC) cell lines. Among these compounds, SKLB0565 displayed noteworthy potency against eight CRC cell lines with IC50 values ranging from 0.012 μM and 0.081 μM. Besides, SKLB0565 inhibited tubulin polymerization, caused G2/M phase cell cycle arrest, depolarized mitochondria and induced cell apoptosis in CRC cells. Furthermore, SKLB0565 suppressed cell migration and disrupted the capillary tube formation of human umbilical vein endothelial cells (HUVECs). Our data clarified that SKLB0565 is a promising anti-tubulin agent for CRC therapy which is worthy of further evaluation.

Lung cancer remains the leading cause of cancer mortality because of highly malignant and metastatic potential. The current status of lung cancer treatment is limited, and more treatment options are needed. Interesting, antipsychotic drugs have been reported to show anti-cancer effects. In this present study, we investigated the anticancer potential of penfluridol (PF), an anti-schizophrenic drug, in lung cancer and its underlying mechanism in vitro and in vivo. In vitro, it could inhibit the viability of various lung cancer cells with G0/G1 phase arrest via increasing the expression level of p21/p27 and decreasing the expression levels of cyclin-CDK complex. Meanwhile, cell-cycle arrest causes DNA repair in the nucleus, which was associated with the upregulation of H2A.X and p-H2A.X. Moreover, PF could also decrease mitochondrial membrane potential and increase reactive oxygen species levels in the lung cancer cells. These results implied that PF might induce the mitochondria-mediated intrinsic apoptosis. In addition, PF inhibits the migration and invasion of lung cancer cells via downregulation of FAK-MMP signaling. In vivo, oral administration of PF at concentration of 10 mg/kg inhibited tumor growth in A549 xenograft model. Notably, PF is an approved drug and the price is exceedingly cheap, so this study demonstrates the potential of PF to treat lung cancer.

Women with aggressive triple-negative breast cancer (TNBC) are at high risk of brain metastasis, which has no effective therapeutic option partially due to the poor penetration of drugs across the blood−brain barrier. Trifluoperazine (TFP) is an approved antipsychotic drug with good bioavailability in brain and had shown anticancer effect in several types of cancer. It drives us to investigate its activities to suppress TNBC, especially the brain metastasis. In this study, we chose three TNBC cell lines MDA-MB-468, MDA-MB-231, and 4T1 to assess its anticancer activities along with the possible mechanisms. In vitro, it induced G0/G1 cell cycle arrest via decreasing the expression of both cyclinD1/CDK4 and cyclinE/CDK2, and stimulated mitochondria-mediated apoptosis. In vivo, TFP suppressed the growth of subcutaneous xenograft tumor and brain metastasis without causing detectable side effects. Importantly, it prolonged the survival of mice bearing brain metastasis. Immunohistochemical analysis of Ki67 and cleaved caspase-3 indicated TFP could suppress the growth and induce apoptosis of cancer cells in vivo. Taken together, TFP might be a potential available drug for treating TNBC with brain metastasis, which urgently needs novel treatment options.

A new series of 3-(((9H-purin-6-yl)amino)methyl)-4,6-dimethylpyridin-2(1H)-one derivatives were designed, synthesized and demonstrated to act as tubulin polymerization inhibitors. These new derivatives showed significant antitumor activities, among which SKLB0533 demonstrated to be the most potent compound, with IC50 values ranging from 44.5 to 135.5 nM against seven colorectal carcinoma (CRC) cell lines. Remarkably, SKLB0533 exhibited no activity against other potential targets, such as 420 kinases and EZH2. Besides, SKLB0533 inhibited tubulin polymerization, arrested the cell cycle at the G2/M phase and induced apoptosis in CRC cells. Furthermore, SKLB0533 suppressed tumour growth in the HCT116 xenograft model without inducing notable major organ-related toxicity, suggesting that SKLB0533 could be used as a promising lead compound for the development of new antitumor agents.

Arresting cell cycle has been one of the most common approaches worldwide in cancer therapy. Specifically, arresting cells in the G2/M phase is a promising therapeutic approach in the battle against lung cancer. In the present study, we demonstrated the anticancer activities and possible mechanism of compound #2714, which can prompt G2/M phase arrest followed by cell apoptosis induction in Lewis lung carcinoma LL/2 cells. In vitro, #2714 significantly inhibited LL/2 cell viability in a concentration- and time-dependent manner while exhibiting few toxicities on non-cancer cells. The mechanism study showed that cell proliferation inhibition due to the treatment with #2714 correlated with G2/M phase arrest and was followed by LL/2 cell apoptosis. The characterized changes were associated with the downregulation of phosphorylated cell division cycle 25C (Cdc25C) and upregulation of p53. Apoptosis-associated activation of cleaved caspase-3 was also detected. Moreover, #2714 strongly attenuated LL/2 cell proliferation by disrupting the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). In vivo, intraperitoneal administration of #2714 (25–100 mg/kg/day) to mice bearing established tumors in xenograft models significantly prevented LL/2 tumor growth (58.1%) without detectable toxicity. Compound #2714 significantly increased apoptosis in LL/2 lung cancer cells in mice models, as observed via terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, and the data from an immunohistochemical analysis showed that #2714 remarkably inhibited the proliferation and angiogenesis of lung cancer in vivo. Taken together, our data suggest that #2714 has a high potential anti-lung cancer efficacy with a pathway-specific mechanism of G2/M phase arrest and subsequent apoptosis induction both in vitro and in vivo; its potential to be an anticancer candidate warrants further investigation.

Hepatocellular carcinoma is the fifth most common cancer and durable responses in conventional treatments are limited so researchers have been devoted to developing new anti-HCC agents. Benzothiazole derivatives are known for various biological activities and have received considerable attention in cancer therapy, hence we designed and synthesized a novel potent benzothiazole compound 2-chloro-N-(2-(2-(2-morpholino-2-oxoethyl)thio)-2,3-dihydrobenzo[d]thiazol-6-yl)acetamide (SKLB826) and further investigated the biological activities against cancer. The results suggested that SKLB826 showed growth inhibition against a broad spectrum of human cancer cells, especially human HCC cell lines, in a dose-dependent manner and induced G2/M phase arrest via down-regulating the CDK1, cyclinA2 and cdc25c protein levels. SKLB826 could also induce apoptosis of HCC cells via decreasing the expression of Bcl-2 and increasing the levels of BAX and cleaved caspase-3, 9. Moreover, after treatment with SKLB826, the change of ROS level and ΔΨm suggested that SKLB826 might induce apoptosis through an intrinsic mitochondrial apoptotic pathway. Furthermore, SKLB826 could suppress tumor growth in the HepG2 xenograft model without inducing any notable major organ-related toxicity, suggesting that SKLB826 may be a potential candidate for HCC therapy.

Benzothiazole derivatives have received considerable attentions for their potencies in cancer therapy. In the present study, we reported that SKLB316, a novel synthesized benzothiazole derivative, exhibits activities to inhibit colorectal and pancreatic cancer in vitro and in vivo by inducing G2/M cell cycle arrest and apoptosis. In vitro, it exhibited significant anti-proliferative activities against human cancer cells derived from different histotypes including the colorectal cancer cell line HCT116 and pancreatic cancer cell line CFPAC-1. We chose these cell lines to study the possible anti-tumor mechanism because they are sensitive to SKLB316 treatment. Flow cytometry assays showed that SKLB316 could induce G2/M cell cycle arrest. Mechanistically, SKLB316 could decrease the activities of cdc2/cyclin B1 complex, including decreasing the synthesis of cyclin B1, cdc2 and cdc25c, while accumulating the levels of phosphorylated cdc2 (Tyr15) and checkpoint kinase 2. SKLB316 could also decrease the level of cyclin E and A2. Moreover, SKLB316 could induce cancer cell apoptosis, which was associated with activation of caspase 9, downregulation of Bcl-2 and upregulation of Bax. SKLB316 could also decrease the mitochondrial membrane potential and induce the generation of reactive oxygen species in cells. The results implied that SKLB316 may induce apoptosis via the mitochondria-mediated apoptotic pathway. Moreover, SKLB316 could suppress the growth of established colorectal and pancreatic cancer tumors in nude mice without causing obvious side effects. TUNEL assays confirmed that SKLB316 could also induce tumor cell apoptosis in vivo. Taken together, these findings demonstrate the potential value of SKLB316 as a novel anti-tumor drug candidate.

Background: Colorectal cancer continues to be one of the most common causes of cancer death, and the poor survival rates and liver metastases at the time of diagnosis urgently need more effective strategy for colorectal cancer treatment. Methods: The activities of N-(5-bromopyridin-2-yl)-2-((6-(2-chloroacetamido)benzo[d]thiazol-2-yl)thio)acetamide (YLT205), which is a novel small molecule compound synthesized by us, were investigated using MTT assay, flow cytometry, western blotting and mice tumor xenograft models. Results: YLT205 induced apoptosis of human colorectal cell lines in a dose-dependent manner. The occurrence of apoptosis was associated with activation of caspases-9 and -3, down-regulation of Bcl-2 and up-regulation of Bax in HCT116 cells. Moreover, YLT205 disrupted mitochondrial membranes and induced the release of cytochrome c into cytosol. Impaired phosphorylation of p44/42 mitogen-activated protein kinase was also observed while the expression of phosphorylated protein kinase B (Akt) was not affected. Furthermore, in HCT116 and SW620 tumor-bearing nude mice models, YLT205 dose-dependently inhibited tumor growth without obvious adverse effects. Immunohistochemistry analyses revealed YLT205 also induced apoptosis and inhibited tumor cell proliferation in vivo. Conclusion: These studies suggested that YLT205 might be a potential drug candidate for human colorectal cancer therapy.

d These authors contributed equally to this work Abstract Background: AZD4547, a small-molecule inhibitor targeting the tyrosine kinase of Fibroblast Growth Factor Receptors (FGFRs), is currently under phase II clinical study for human subjects having breast cancer, while the underlying mechanism remains elusive. The aim of this study is to explore the potential mechanism by which AZD4547 inhibits breast tumor lung metastases at the level of the tumor microenvironment. Methods: First, through in vitro experiments, we investigated the efficacy of the FGFRs inhibitor AZD4547 on 4T1 tumor cells for their proliferation, apoptosis, migration, and invasion. Second, by in vivo animal experiments, we evaluated the effects of AZD4547 on tumor growth and lung metastases in 4T1 tumor-bearing mice. Finally, we examined the impact of AZD4547 on the infiltration of myeloid-derived suppressor cells (MDSCs) in lung, spleens, peripheral blood and tumor. Results: Through this study we found that AZD4547 could efficiently suppress tumor 4T1 cells through restraining their proliferation, blocking migration and invasion, and inducing apoptosis in vitro. In animal model we also demonstrated that AZD4547 was able to inhibit tumor growth and lung metastases, consistent with the decreased MDSCs accumulation in the tumor and lung tissues, respectively. Moreover, the reduced number of MDSCs in peripheral blood and spleens were also observed in the AZD4547-treated mice. Importantly, through the AZD4547 treatment, the CD4 + and CD8 + T-cells were significantly increased in tumor and spleens. Conclusion: Our studies showed that AZD4547 can inhibit breast cancer cell proliferation, induce its apoptosis and block migration and invasion in vitro and suppress tumor growth and lung metastases by modulating the tumor immunologic microenvironment in vivo.

Background and Purpose Targeted chemotherapy using small-molecule inhibitors of angiogenesis and proliferation is a promising strategy for cancer therapy. Experimental Approach YL529 was developed via computer-aided drug design, de novo synthesis and high-throughput screening. The biochemical, pharmacodynamic and toxicological profiles of YL529 were investigated using kinase and cell viability assays, a mouse tumour cell-containing alginate bead model, a zebrafish angiogenesis model and several human tumour xenograft models in athymic mice. Key Results In vitro, YL529 selectively inhibited the activities of VEGFR2/VEGFR3 and serine/threonine kinase RAF kinase. YL529 inhibited VEGF165-induced phosphorylation of VEGFR2, as well as the proliferation, migration, invasion and tube formation of human umbilical vascular endothelial cells. It also significantly blocked vascular formation and angiogenesis in the zebrafish model. Moreover, YL529 strongly attenuated the proliferation of A549 cells by disrupting the RAF/mitogen-activated protein (MAP) or extracellular signal-regulated kinase (Erk) kinase (MEK) kinase kinase/MAPK pathway. Oral administration of YL529 (37.5–150 mg−1·kg−1·day−1) to nude mice bearing established tumour xenografts significantly prevented the growth (60–80%) of A549, SPC-A1, A375, OS-RC-2 and HCT116 tumours without detectable toxicity. YL529 markedly reduced microvessel density and increased tumour cell apoptosis in the tumours formed in mice inoculated with the lung cancer cells, SPC-A1 and A549, and the colon carcinoma cells, HCT116. Conclusions and Implications YL529, an orally active multikinase inhibitor, shows therapeutic potential for solid tumours, and warrants further investigation as a possible anticancer agent.

Melanoma is the most serious type of skin cancer because it is highly frequency of drug resistance and can spread earlier and more quickly than other skin cancers. The objective of this research was to investigate the anticancer effects of cryptotanshinone on human melanoma cells in vitro, and explored its mechanisms of action. Our results have shown that cryptotanshinone could inhibit cell proliferation in human melanoma cell lines A2058, A375, and A875 in a dose- and time-dependent manner. In addition, flow cytometry assay showed that cryptotanshinone inhibited the proliferation of human melanoma cell line A375 by blocking cell cycle progression in G2/M phase and inducing apoptosis in a concentration-dependent manner. Moreover, western blot analysis indicated that the occurrence of its apoptosis was associated with upregulation of cleaved caspases-3 and pro-apoptotic protein Bax while downregulation of anti-apoptotic protein Bcl-2. Meanwhile, cryptotanshinone could decrease the levels of reactive oxygen species (ROS). Furthermore, cryptotanshinone also blocked A375 cell migration and invasion in vitro which was associated with the downregulation with MMP-9. Taken together, these results suggested that cryptotanshinone might be a potential drug in human melanoma treatment by inhibiting proliferation, inducing apoptosis via ROS-mitochondrial apoptotic pathway and blocking cell migration and invasion.

Melanoma is a highly malignant neoplasm of melanocytes with considerable metastatic potential and drug resistance, explaining the need for new candidates that inhibit tumor growth and metastasis. The signal transducer and activator of the transcription 3 (Stat3) signaling pathway plays an important role in melanoma and has been validated as promising anticancer target for melanoma therapy. In this study, nifuroxazide, an antidiarrheal agent identified as an inhibitor of Stat3, was evaluated for its anti-melanoma activity in vitro and in vivo. It had potent anti-proliferative activity against various melanoma cell lines and could induce G2/M phase arrest and cell apoptosis. Moreover, nifuroxazide markedly impaired melanoma cell migration and invasion by down-regulating phosphorylated-Src, phosphorylated-FAK and expression of matrix metalloproteinase (MMP) -2, MMP-9 and vimentin. It also significantly inhibited tumor growth without obvious side effects in the A375-bearing mice model by inducing apoptosis and reducing cell proliferation and metastasis. Notably, nifuroxazide significantly inhibited pulmonary metastases, which might be associated with the decrease of myeloid-derived suppressor cells (MDSCs). These findings suggested that nifuroxazide might be a potential agent for inhibiting the growth and metastasis of melanoma.

Enhancer of zeste homolog 2 (EZH2) is a candidate oncogenic driver due to its prevalent overexpression and aberrant repression of tumor suppressor genes in diverse cancers. Therefore, blocking EZH2 enzyme activity may present a valid therapeutic strategy for the treatment of cancers with EZH2 overexpression including breast cancers. Here, we described ZLD1039 a potent, highly selective, and orally bioavailable small molecule inhibitor of EZH2, which inhibited breast tumor growth and metastasis. ZLD1039 considerably inhibited EZH2 methyltransferase activity with nanomolar potency, decreased global histone-3 lysine-27 (H3K27) methylation, and reactivated silenced tumor suppressors connected to increased survival of patients with breast cancer. Comparable to conditional silencing of EZH2, its inhibition by ZLD1039 decreased cell proliferation, cell cycle arrest, and induced apoptosis. Comparably, treatment of xenograft-bearing mice with ZLD1039 led to tumor growth regression and metastasis inhibition. These data confirmed the dependency of breast cancer progression on EZH2 activity and the usefulness of ZLD1039 as a promising treatment for breast cancer.

We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro . We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro , and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G 0 /G 1 phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G 0 /G 1 phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.

A series of novel benzothiazole-2-thiol derivatives were synthesized and their structures determined by 1H-NMR, 13C-NMR and HRMS (ESI). The effects of all compounds on a panel of different types of human cancer cell lines were investigated. Among them, pyridinyl-2-amine linked benzothiazole-2-thiol compounds 7d, 7e, 7f and 7i exhibited potent and broad-spectrum inhibitory activities. Compound 7e displayed the most potent anticancer activity on SKRB-3 (IC(50) = 1.2 nM), SW620 (IC(50) = 4.3 nM), A549 (IC(50) = 44 nM) and HepG2 (IC(50) = 48 nM) and was found to induce apoptosis in HepG2 cancer cells.

Inducing apoptosis is a promising therapeutic approach to overcome cancer. Here we described that a novel synthesized compound, 3-amino-N-(4-chlorobenzyl)-6-(3-methoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide (SKLB703), exhibits antitumor activity via inducing apoptosis both invitro and invivo. Our results showed that SKLB703 inhibited the proliferation of a panel of human cancer cell lines, and human hepatocellular carcinoma cell line HepG2 was the most sensitive. The proliferation inhibitory effect of SKLB703 was associated with its apoptosis-inducing effect by activating caspase-3 and caspase-9 rather than caspase 8. Exposure of HepG2 to SKLB703 also resulted in Bax upregulation, Bcl-2 downregulation, cytochrome c release and mitochondrial transmembrane potential change in mitochondrial apoptotic pathway. Moreover, the decrease of phosphorylated p 44/42 mitogen-activated protein kinase and phosphorylated Akt was observed. SKLB703 suppressed the growth of established tumors in xenograft models in mice, whereas no toxicity was exhibited. TUNAL analysis showed that SKLB703 induced HepG2 tumor apoptosis. Taken together, the present study demonstrates that SKLB730 exhibits its antitumor activity through inducing apoptosis via mitochondrial apoptotic pathway. Its potential to be a candidate of anticancer agent is worth being further investigated.

In a cell-based screen of novel anticancer agents, the hit compound 1a which bears a pyrazolo[3,4-d]pyrimidine scaffold exhibited high inhibitory activity against a panel of four different types of tumor cell lines. In particular, the IC₅₀ for A549 cells was 2.24 µM, compared with an IC₅₀ of 9.20 µM for doxorubicin, the positive control. Four synthetic routes of the key intermediate 3 of 1a were explored and 1a was prepared via route D on the gram scale for further research. Two analogs of 1a were synthesized and their preliminary structure-activity relationships were studied. Flow cytometric analysis revealed that compound 1a could significantly induce apoptosis in A549 cells in vitro at low micromolar concentrations. These results suggest that the target compound 1a and its analogs with the pyrazolo[3,4-d]pyrimidine scaffold might potentially constitute a novel class of anticancer agents, which requires further studies.

Novel thienopyridine derivatives 1b-1r were synthesized, based on a hit compound 1a that was found in a previous cell-based screening of anticancer drugs. Compounds 1a-1r have the following features: (1) their anticancer activity in vitro was first reported by our group. (2) The most potent analog 1g possesses hepatocellular carcinoma (HCC)-specific anticancer activity. It can specifically inhibit the proliferation of the human hepatoma HepG2 cells with an IC(50) value of 0.016μM (compared with doxorubicin as a positive control, whose IC(50) was 0.37μM). It is inactive toward a panel of five different types of human cancer cell lines. (3) Compound 1g remarkably induces G(0)/G(1) arrest and apoptosis in HepG2 cells in vitro at low micromolar concentrations. These results, especially the HCC-specific anticancer activity of 1g, suggest their potential in targeted chemotherapy for HCC.

EZH2 (Enhancer of zeste homolog 2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which is involved in repressing gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation. EZH2 overexpression is implicated in tumorigenesis and has been a candidate oncogene in several tumor types. Recently, point mutations of EZH2 at Tyr641 and Ala677 were identified in diffuse large B cell lymphoma and follicular lymphoma, where they drive H3K27 hypertrimethylation and cancer progression. Here, we reported a novel, highly potent and selective small molecule inhibitor of EZH2, ZLD10A, which inhibited wild-type and mutant versions of EZH2 with nanomolar potency and had greater than 1000-fold selectivity against 10 other histone methyltransferases. Our results have shown that the compound suppressed global H3K27 methylation and cause the anti-proliferation effects in a concentration- and time-dependent manner in DLBCL cell lines. These results demonstrated that ZLD10A, as a novel EZH2 inhibitor, could be a potential promising agent for the treatment of EZH2 mutant lymphoma.

Colorectal carcinoma (CRC) is the one of the most common cancers with considerable metastatic potential, explaining the need for new drug candidates that inhibit tumor metastasis. The signal transducers and activators of the transcription 3 (Stat3) signaling pathway has an important role in CRC and has been validated as a promising anticancer target for CRC therapy. In the present study, we report our findings on nifuroxazide, an antidiarrheal agent identified as an inhibitor of Stat3. Our studies showed that nifuroxazide decreased the viability of three CRC cell lines and induced apoptosis of cancer cells in a concentration-dependent manner. Moreover, western blot analysis demonstrated that the occurrence of its apoptosis was correlated with the activation of Bax and cleaved caspase-3, and decreased the expression of Bcl-2. In addition, nifuroxazide markedly impaired CRC cell migration and invasion by downregulating phosphorylated-Stat3Tyr705, and also impaired the expression of matrix metalloproteinases (MMP-2 and MMP-9). Furthermore, our studies showed that nifuroxazide also significantly inhibited the tumor metastasis in lung and abdomen metastasis models of colon cancer. Meanwhile, nifuroxazide functionally reduced the proliferation index, induced tumor apoptosis and impaired metastasis. Notably, nifuroxazide reduced the number of myeloid-derived suppressor cells in the blood, spleens and tumors, accompanied by the increased infiltration of CD8+ T cells in the tumors. Importantly, a marked decrease in the number of M2-type macrophages in tumor in the abdomen metastasis model was also observed. Taken together, our results indicated that nifuroxazide could effectively inhibit tumor metastasis by mediating Stat3 pathway and it might have a therapeutic potential for the treatment of CRC.

Background: Breast cancer is the leading cause of cancer death among women worldwide and metastasis is the major cause of treatment failure. Thus, new treatment options for breast cancer, especially, drugs which could prevent metastasis, are pressingly needed. Methods: In the present study, we designed and synthesized a novel cinnamide derivative, (E)-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (YLT26), which displayed potent inhibitory effects on breast cancer cells. The cell viability, apoptosis-inducing effect and reactive oxygen species (ROS) production were examined in 4T1 cells following treatment with YLT26. Meanwhile, apoptosis-related proteins levels were determined by western blotting. Finally, we evaluated the effects of YLT26 on breast tumor growth, lung metastases in vivo and the infiltration of myeloid-derived suppressor cells (MDSCs) in lung tissue. Results: Our results showed that the proliferation inhibitory effects of YLT26 were correlated with its apoptosis-inducing effect. Exposure to YLT26 induced mitochondrial transmembrane potential (∆Ψm) change, activated caspase-9, and downregulated the Bcl-2 expression, as well as enhanced ROS accumulation in 4T1 cells. Moreover, YLT26 significantly inhibited tumor growth without obvious side effects in the 4T1 tumor-bearing mice model. Immunohistochemistry analyze revealed YLT26 also induced apoptosis in vivo. More importantly, YLT26 also significantly inhibited lung metastases, which may be associated with the reduction of MDSCs. Conclusion: The present study suggested that YLT26 could inhibit breast cancer cells proliferation via ROS-mitochondrial apoptotic pathway, delay breast tumor progression, and suppress lung metastases by impacting on the immunologic microenvironment in vivo.

Breast carcinoma is the most common female cancer with considerable metastatic potential. Discovery of new therapeutic approaches for treatment of metastatic breast cancer is still needed. Here, we reported our finding with niclosamide, an FDA approved anthelmintic drug. The potency of niclosamide on breast cancer was assessed in vitro and in vivo. In this investigation, we found that niclosamide showed a dramatic growth inhibition against breast cancer cell lines and induced apoptosis of 4T1 cells in a dose-dependent manner. Further, Western blot analysis demonstrated the occurrence of its apoptosis was associated with activation of Cleaved caspases-3, down-regulation of Bcl-2, Mcl-1 and Survivin. Moreover, niclosamide blocked breast cancer cells migration and invasion, and the reduction of phosphorylated STAT3(Tyr705), phosphorylated FAK(Tyr925) and phosphorylated Src(Tyr416) were also observed. Furthermore, in our animal experiments, intraperitoneal administration of 20 mg/kg/d niclosamide suppressed 4T1 tumor growth without detectable toxicity. Histological and immunohistochemical analyses revealed a decrease in Ki67-positive cells, VEGF-positive cells and microvessel density (MVD) and an increase in Cleaved caspase-3-positive cells upon niclosamide. Notably, niclosamide reduced the number of myeloid-derived suppressor cells (MDSCs) in tumor tissues and blocked formation of pulmonary metastases. Taken together, these results demonstrated that niclosamide may be a promising candidate for breast cancer.

Small-molecule inhibitors are an attractive therapeutic approach for most types of human cancers. SKLB-163, a novel benzothiazole-2-thiol derivative, was developed via computer-aided drug design and de novo synthesis. MTT assay showed it had potent anti-proliferative activity on various human cancer cells. Treatment of cancer cells with SKLB-163 induced obvious apoptosis and inhibited proliferation in vitro. SKLB-163 administered p.o. showed a marked antitumor activity in vivo. Proteomic techniques were employed to identify possible drug target proteins. The data showed molecular mechanism of action might be involved in downregulation of RhoGDI, which finally contributed to increased apoptosis and inhibited proliferation. These findings provided the potential value of SKLB-163 as a novel candidate antitumor drug.

Aberrant fibroblast growth factor (FGF) and FGF receptor (FGFR) system have been associated with breast cancer. The objectives of our study were to investigate the effects and mechanisms of FGFR inhibition on tumor growth and metastasis on breast cancer. Our studies showed that the FGFR inhibitor PD173074 decreased the viability of several human breast cancer cells, as well as 4T1 murine mammary tumor cells. Therefore, we chose 4T1 cells to study PD173074's antitumor mechanism. Flow cytometry showed that PD173074 induced 4T1 cell apoptosis in a concentration-dependent manner. Western blot demonstrated that PD173074-induced apoptosis was correlated with the inhibition of Mcl-1 and survivin. Moreover, PD173074 also significantly increased the ratio of Bax/Bcl-2. PD173074 could also block 4T1 cell migration and invasion in vitro. In 4T1 tumor-bearing mice, PD173074 significantly inhibited tumor growth without obvious side effects. Meanwhile, PD173074 functionally reduced microvessel density and proliferation index and induced tumor apoptosis. Importantly, we found that FGFR inhibition by PD173074 reduced myeloid-derived suppressor cells (MDSCs) in the blood, spleens and tumors, accompanied by the increased infiltration of CD4(+) and CD8(+) T cells in the spleens and tumors. Furthermore, PD173074 significantly inhibited breast tumor metastasis to the lung of inoculated 4T1 breast cancer cells, which was accompanied by a reduction in MDSCs. Our findings suggested that FGFR inhibition could delay breast tumor progression, impair lung metastasis and break immunosuppression by effecting on tumor microenvironment, which may provide a promising therapeutic approach for breast cancer patient.

Inducing apoptosis is a promising therapeutic approach to overcome cancer. In this study, 30 compounds were synthesized and evaluated for their antiproliferative activity against three tumor cell lines in vitro: A875, H460 and Hela cancer cells by the MTT assay. The most potent analogue 7a , a novel compound was first reported by our group, inhibited the proliferation of A875 cells with an IC 50 value of 98 nM. Flow cytometry analysis and morphological analysis suggested that compound 7a had potential anticancer efficacy via G 2 /M cell cycle arrest, which could be attributed to its proliferation and apoptosis, and also in a concentration-dependent manner. The SAR analysis indicated that the substituents R 2 played a crucial role in the antiproliferation activity.

We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro. We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro, and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G(0)/G(1) phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G(0)/G(1) phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.

Hepatocellular carcinoma is one of the most common cancers in worldwide. We previously reported a novel thienopyridine derivative 3-amino-6-(3,4-dichlorophenyl) thieno[2,3-b]pyridine-2-carboxamide (SKLB70359) which possesses anticancer activity against hepatocellular carcinoma. In present study, we further investigated its anticancer activity and possible mechanism. The SKLB70359 treatment decreased the viability of a panel of hepatocellular carcinoma cell lines in a concentration- and time-dependent manner with IC(50) 0.4 ~ 2.5 μM. The mechanism study showed that SKLB70359 induced G0/G1 cell cycle arrest and then led to apoptotic cell death of HepG2 cell. The SKLB70359 induced G0/G1 cell cycle arrest was characterized by down-regulation of cyclin-dependent kinase 2 (CDK2), CDK4, CDK6 expression and up-regulation of p53, p21(WAF1). Activating of caspase-3 and caspase-9 was also observed. Meanwhile, proliferation inhibitory effect of SKLB70359 was associated with decreased level of phosphorylated p44/42 mitogen activated protein kinase (p44/42 MAPK) and phosphorylated retinoblastoma protein (Rb). Moreover, SKLB70359 exhibit less toxicity to non-cancer cells than tumor cells. In conclusion, the findings in this study suggested that SKLB70359 have potential anticancer efficacy via G0/G1 cell cycle arrest and apoptosis induction. Its potential to be a candidate of anticancer agent is worth being further investigated.

A series of novel benzothiazole-2-thiol derivatives were synthesized, and their anti-proliferative activities on HepG2 and MCF-7 cells were investigated. Most compounds had inhibitory effects on cell growth, and some of them were more effective than cisplatin. Compounds 6m and 6t displayed good inhibitory activities against a panel of different types of human cancer cell lines, with IC(50) values in the low micromolar range. Further biological evaluation indicated that 6m induced apoptosis in HepG2 cancer cells. Structure-activity relationships were also proposed.