Preclinical safety profile of disitamab vedotin：a novel anti-HER2 antibody conjugated with MMAE.

The HER2 pathway plays a pivotal role in cell proliferation and differentiation, while the receptor overexpression caused by amplification of HER2 gene is associated with the growth of several tumors. Previously published clinical trials have demonstrated that antibody-conjugated drugs (ADCs) remarkably improved clinical effects compared with antibodies alone for the same target. In order to provide more effective drugs, we developed Disitamab vedotin based on ADC. The antibody part was a humanized monoclonal antibody targeting HER2, the small molecule toxin was monomethyl auristatin E (MMAE), a synthetic antineoplastic agent. A protease cleavable linker covalently attached MMAE to the antibody. In this study, we characterized the toxicity profile of Disitamab vedotin through single- and repeat-dose toxicity studies in monkeys. The toxicities of small molecules and naked antibody (Disitamab) were also assessed in these studies. Monkeys were well tolerated with Disitamab vedotin at doses of 6 mg/kg, while equivalent MMAEs resulted in severe myelosuppression. This finding proves that ADCs improve the therapeutic effect. In addition, the safety profiles of Disitamab vedotin and MMAE were similar and consistent with the activation mechanism of MMAE. Toxicology finding included bone marrow/hematology toxicity and lymphoid organ toxicity, while no significant toxicity was observed in animals treated with naked antibody. These side effects were found to be consistent with data acquired from clinical phase I/II patients treated with Disitamab vedotin.

Astrocytic transactivation by alpha2A-adrenergic and 5-HT2B serotonergic signaling.

EGF receptor transactivation has been known for more than ten years. It is a signal pathway in which a G-protein-coupled receptor (GPCR) signal leads to release of a growth factor, which in turn activates the EGF receptor-tyrosine kinase in the same or adjacent cells. Astrocytes express a number of GPCRs and play key roles in brain function. Astrocytic transactivation is of special interest, since its autocrine effect may regulate gene expression and alter cell functions in the cells themselves and its paracrine effect may provide additional opportunities for cross-talk between astrocytes and their neighbors, such as neurons. The signal pathways of EGF transactivation are complicated. This does not only apply to the pathways leading to shedding of growth factor(s), but also to the downstream signal pathways of the EGF receptor, i.e., MAPK and PI3K. The latter may vary according to the type of growth factor released, the sites of tyrosine phosphorylation on the EGF receptor, and the duration of the phosphorylation. Using primary cell cultures we have found that dexmedetomidine, a specific alpha(2)-adrenergic receptor, induced shedding of HB-EGF from astrocytes, which in turn transactivated EGF receptors and stimulated astrocytic c-Fos and FosB expression. At the same time released HB-EGF protected neurons from injury caused by H(2)O(2). We have also confirmed dexmedetomidine transactivation in the brain in vivo. EGF transactivation by 5-HT(2B) receptor stimulation was responsible for up-regulation of cPLA(2) in astrocytes by fluoxetine, an antidepressant and inhibitor of the serotonin transporter, which also is a specific 5-HT(2B) agonist.

Inhibition of pancreatic cancer cell growth by cucurbitacin B through modulation of signal transducer and activator of transcription 3 signaling.

OBJECTIVES: Pancreatic cancer is a serious disease worldwide for its high mortality. Gemcitabine has become the frontline option for the treatment of this disease since its approval. However, resistance to the drug has been on the rise in recent years. Searching for other chemotherapeutic agents therefore has attracted much attention. Cucurbitacin B (CuB) is a member of the triterpenoid family and has shown inhibitory effect on various cancer cells. In this study, we have assessed the effect of CuB on pancreatic cancer cells. METHODS: The growth of human pancreatic cancer cells (PANC-1) was monitored using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell cycle distribution and apoptosis were evaluated with fluorescence-activated cell sorter and fluorescent microscopy. Western blot was used to determine the expression of relevant genes including phosphorylated signal transducer and activator of transcription 3 (pSTAT3), STAT3, p53, p21, Bcl-2, survivin, and caspase 3. RESULTS: Our results showed that CuB can inhibit the growth of PANC-1 cells in a dose- and time-dependent manner, resulting in accumulation of G2/M phase cells and apoptosis. Furthermore, CuB treatment inhibited STAT3 phosphorylation, activated caspase 3, up-regulated the expression of p53 and p21, and down-regulated the expression of Bcl-2 and survivin. CONCLUSIONS: Our results suggested that CuB may provide an effective regimen for the treatment of pancreatic cancers.

Inhibitory effect of cucurbitacin E on pancreatic cancer cells growth via STAT3 signaling.

PURPOSE: Pancreatic cancer has been a serious disease worldwide for its high mortality. Cucurbitacin E is a member of triterpenoid family isolated from plants showing antiproliferative activity on various cancer cells. In this study, we have explored whether cucurbitacin E also has an anti-tumor effect on pancreatic cancer cells. METHODS: Human pancreatic cancer cells PANC-1 were used to explore the effect and possible mechanisms of cucurbitacin E on cell cycle progression, apoptosis and proliferation. RESULTS: Cucurbitacin E has inhibited the growth of PANC-1 cells in a dose- and time-dependent manner, and has caused accumulation of cells at the G(2)/M phase as well as apoptosis. Western blotting also showed that cucurbitacin E treatment can inhibit STAT3 phosphorylation while upregulate p53 expression. CONCLUSIONS: Our results suggested that cucurbitacin E may be an effective regimen for the chemotherapy of pancreatic cancer.

[Inhibitory effect of cucurbitacin E on the proliferation of ovarian cancer cells and its mechanism].

BACKGROUND AND OBJECTIVE: There are various biological activities of cucurbitacin E (CuE), including antitumor effect, anti-chemical carcino-genesis, liver protection, and enhancement of the immunity, and so on. This study was to investigate the effect of CuE on proliferation inhibition and apoptosis induction of ovarian cancer ES-2 cells, and to explore the mechanism. METHODS: ES-2 cells were treated with different concentrations of CuE for 24, 48, and 72 h, respectively. Cell proliferation was tested by MTT assay. The morphologic changes and apoptosis were observed under inverted microscope and fluorescent microscope. Cell cycle distribution was evaluated with flow cytometry. The expression of p-STAT3 was determined by Western blot. RESULTS: The number of ES-2 significantly decreased as the concentration of CuE increased or the time prolonged. Flow cytometry analysis showed that the ratio of ES-2 cells treated 1 micromol/L CuE for 24 h increased both in S phase [from (10.55+/-0.91)% to ( 16.31 +/- 4.61) % ] and in G(2)/M phase [from (18.53+/-1.43)% to (58.34 +/- 5.77)%], while decreased in G(1) phase [from (73.13 +/-4.70)% to (23.12 +/- 5.45)%] (P<0.05). The marked morphological changes of cell apoptosis were clearly observed in ES-2 cells treated with CuE. CuE inhibited the STAT3 phosphorylation in ES-2 cell in a dose- dependent manner. CONCLUSION: CuE can inhibit ES-2 proliferation and induce apoptosis and cell cycle arrest, which may be related to the decreased expression of the intracellular STAT3 phosphorylation.

Targeted constitutive activation of signal transducer and activator of transcription 3 in human hepatocellular carcinoma cells by cucurbitacin B.

PURPOSE: To determine the effect of cucurbitacin B on human hepatocellular carcinoma cell growth and apoptosis, and to explore the potential mechanisms. METHODS: In vitro viability of human hepatocellular carcinoma cell line (HepG2) was investigated using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Morphologic changes of cells were evaluated through light microscopy. Cell cycle distribution was evaluated with flow cytometry following PI staining. Apoptosis was evaluated respectively with flow cytometry and fluorescent microscopy following Annexin V-FITC/PI and Hoechst 33258 staining. Western blot assays were performed to determine the expression of pSTAT3 and Bcl-2. Finally, in vivo effect of cucurbitacin B on the growth of HepG2 cells was determined in nude mice. RESULTS: The MTT assay showed that cucurbitacin B inhibited HepG2 cell viability in a dose and time-dependent manner. Cucurbitacin B treatment resulted in accumulation of cells at the S phase of cell cycle as well as apoptosis. Marked morphological changes, including condensation of chromatin, nuclear fragmentation and apoptotic bodies were clearly shown on Hoechst 33258 staining. Western blot showed that cucurbitacin B inhibited STAT3 phosphorylation and down-regulated the expression of Bcl-2. Growth of HepG2 tumor in nude mice was also inhibited by cucurbitacin B. CONCLUSION: Our results suggest that cucurbitacin B may have a therapeutic value in suppressing the growth of human hepatocellular carcinoma. The mechanism may be attributable to the suppression of STAT3 phosphorylation.