Tinghong Ye

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.

Cationic nanocarriers induce cell necrosis through impairment of Na+/K+-ATPase and cause subsequent inflammatory response

Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical applications. The mechanism underlying their toxicity, such as the induction of inflammatory response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na+ overload was found to accompany the cell death. Depletion of Na+ in culture medium or pretreatment of cells with the Na+/K+-ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na+/K+-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na+/K+-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na+/K+-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery.

Nifuroxazide induces apoptosis and impairs pulmonary metastasis in breast cancer model

Breast carcinoma is the most common female cancer with considerable metastatic potential. Signal transducers and activators of the transcription 3 (Stat3) signaling pathway is constitutively activated in many cancers including breast cancer and has been validated as a novel potential anticancer target. Here, we reported our finding with nifuroxazide, an antidiarrheal agent identified as a potent inhibitor of Stat3. The potency of nifuroxazide on breast cancer was assessed in vitro and in vivo. In this investigation, we found that nifuroxazide decreased the viability of three breast cancer cell lines and induced apoptosis of cancer cells in a dose-dependent manner. In addition, western blot analysis demonstrated that the occurrence of its apoptosis was associated with activation of cleaved caspases-3 and Bax, downregulation of Bcl-2. Moreover, nifuroxazide markedly blocked cancer cell migration and invasion, and the reduction of phosphorylated-Stat3Tyr705, matrix metalloproteinase (MMP) MMP-2 and MMP-9 expression were also observed. Furthermore, in our animal experiments, intraperitoneal administration of 50 mg/kg/day nifuroxazide suppressed 4T1 tumor growth and blocked formation of pulmonary metastases without detectable toxicity. Meanwhile, histological and immunohistochemical analyses revealed a decrease in Ki-67-positive cells, MMP-9-positive cells and an increase in cleaved caspase-3-positive cells upon nifuroxazide. Notably, nifuroxazide reduced the number of myeloid-derived suppressor cell in the lung. Our data indicated that nifuroxazide may potentially be a therapeutic agent for growth and metastasis of breast cancer.

Synthesis and biological evaluation of novel benzothiazole-2-thiol derivatives as potential anticancer agents.

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 (IC50 = 1.2 nM), SW620 (IC50 = 4.3 nM), A549 (IC50 = 44 nM) and HepG2 (IC50 = 48 nM) and was found to induce apoptosis in HepG2 cancer cells.

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.

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts☆

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p<0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.

Novel thermosensitive hydrogel for preventing formation of abdominal adhesions

Adhesions can form after almost any type of abdominal surgery. Postoperative adhesions can be prevented by improved surgical techniques, such as reducing surgical trauma, preventing ischemia, and avoiding exposure of the peritoneal cavity to foreign materials. Although improved surgical techniques can potentially reduce formation of adhesions, they cannot be eliminated completely. Therefore, finding more effective methods to prevent postoperative adhesions is imperative. Recently, we found that a novel thermosensitive hydrogel, ie, poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCEC) had the potential to prevent postoperative adhesions. Using the ring-opening polymerization method we prepared a PCEC copolymer which could be dissolved and assembled at 55°C into PCEC micelles with mean size of 25 nm. At body temperature, a solution containing PCEC micelles could convert into a hydrogel. The PCEC copolymer was biodegradable and had low toxicity in vitro and in vivo. We found that most animals in a hydrogel-treated group (n = 10) did not develop adhesions. In contrast, 10 untreated animals developed adhesions that could only be separated by sharp dissection (P < 0.001). The hydrogel could adhere to peritoneal wounds and degraded gradually over 7–9 days, transforming into a viscous fuid that was completely absorbed within 12 days. The injured parietal and visceral peritoneum remesothelialized over about seven and nine days, respectively. This study confirms that PCEC hydrogel has potential application in the prevention of postoperative adhesions.