Ningyu Wang

The Anthelmintic Drug Niclosamide Induces Apoptosis, Impairs Metastasis and Reduces Immunosuppressive Cells in Breast Cancer Model

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 STAT3Tyr705, phosphorylated FAKTyr925 and phosphorylated SrcTyr416 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.

Inhibition of FGFR signaling by PD173074 improves antitumor immunity and impairs breast cancer metastasis.

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.

Synthesis and structure–activity relationships evaluation of benzothiazinone derivatives as potential anti-tubercular agents

N-Alkyl and heterocycle substituted 1,3-benzothiazin-4-one (BTZ) derivatives were synthesized. The anti-mycobacterial activities of these compounds were evaluated by determination of minimal inhibitory concentration (MIC) for Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Rv. It was found that an extended or branched alkyl chain analog could enhance the potency, and activities of N-alkyl substituted BTZs were not affected by either nitro or trifluoromethyl at 6-position. Trifluoromethyl plays an important role in maintaining anti-tubercular activity in the piperazine or piperidine analogs. Compound 8o, which contains an azaspirodithiolane group, showed a MIC of 0.0001 μM against M. tuberculosis H37Rv, 20-fold more potent than BTZ043 racemate. These results suggested that the volume and lipophilicity of the substituents were important in maintaining activity. In addition, compound 8o was nontoxic to Vero cells and orally bioavailable in a preliminary pharmacokinetics study.

A Novel Cinnamide YLT26 Induces Breast Cancer Cells Apoptosis via ROS- Mitochondrial Apoptotic Pathway in Vitro and Inhibits Lung Metastasis in Vivo

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.

Discovery of imidazo[2,1-b]thiazole HCV NS4B inhibitors exhibiting synergistic effect with other direct-acting antiviral agents.

The design, synthesis, and SAR studies of novel inhibitors of HCV NS4B based on the imidazo[2,1-b]thiazole scaffold were described. Optimization of potency with respect to genotype 1b resulted in the discovery of two potent leads 26f (EC50 = 16 nM) and 28g (EC50 = 31 nM). The resistance profile studies revealed that 26f and 28g targeted HCV NS4B, more precisely the second amphipathic α helix of NS4B (4BAH2). Cross-resistance between our 4BAH2 inhibitors and other direct-acting antiviral agents targeting NS3/4A, NS5A, and NS5B was not observed. For the first time, the synergism of a series of combinations based on 4BAH2 inhibitors was evaluated. The results demonstrated that our 4BAH2 inhibitor 26f was synergistic with NS3/4A inhibitor simeprevir, NS5A inhibitor daclatasvir, and NS5B inhibitor sofosbuvir, and it could also reduce the dose of these drugs at almost all effect levels. Our study suggested that favorable effects could be achieved by combining 4BAH2 inhibitors such as 26f with these approved drugs and that new all-oral antiviral combinations based on 4BAH2 inhibitors were worth developing to supplement or even replace current treatment regimens for curing HCV infection.

A novel small-molecule YLT205 induces apoptosis in human colorectal cells via mitochondrial apoptosis pathway in vitro and inhibits tumor growth in vivo.

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.

SKLB316, a novel small-molecule inhibitor of cell-cycle progression, induces G2/M phase arrest and apoptosis in vitro and inhibits tumor growth in vivo

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.

Synthesis and antitubercular evaluation of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives

Tuberculosis (TB) remains a major human health problem. New therapeutic antitubercular agents are urgent needed to control the global tuberculosis pandemic. We synthesized a new series of 4-carbonyl piperazine substituted 1,3-benzothiazin-4-one derivatives and evaluated their anti-mycobacterial activities against Mycobacterium tuberculosis H37Ra as well as their druggabilities. The results showed that most of these derivatives, especially the compounds with simple alkyl side chains, exhibited good antitubercular activities and favorable aqueous solubilities with no obvious cytotoxicity. It suggested that the 4-carbonyl piperazine substituents in benzothiazinone scaffold were well tolerated, in which the compound 8h, with an antitubercular activity of MIC 0.008 μM, exhibited an excellent aqueous solubility of 104 μg/mL, which was 100-fold better than the potent DprE1 inhibitor Comp.1 (BTZ038), also more soluble than PBTZ169.

Discovery and structure–activity relationships study of novel thieno[2,3-b]pyridine analogues as hepatitis C virus inhibitors

Current treatment for hepatitis C is barely satisfactory, there is an urgent need to develop novel agents for combating hepatitis C virus infection. This study discovered a new class of thieno[2,3-b]pyridine derivatives as HCV inhibitors. First, a hit compound characterized by a thienopyridine core was identified in a cell-based screening of our privileged small molecule library. And then, structure activity relationship study of the hit compound led to the discovery of several potent compounds without obvious cytotoxicity in vitro (12c, EC50=3.3μM, SI >30.3, 12b, EC50=3.5μM, SI >28.6, 10l, EC50=3.9μM, SI >25.6, 12o, EC50=4.5μM, SI >22.2, respectively). Although the mechanism of them had not been clearly elucidated, our preliminary optimization of this class of compounds had provided us a start point to develop new anti-HCV agents.

Inhibition of Stat3 signaling pathway by nifuroxazide improves antitumor immunity and impairs colorectal carcinoma metastasis

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.