In vitro refolding and functional analysis of polyhistidine-tagged Buthus martensii Karsch antitumor-analgesic peptide produced in Escherichia coli.

OBJECTIVES: To identify an efficient in vitro refolding method to generate highly active His6-tagged scorpion toxin antitumor-analgesic peptide (AGAP) isolated from Escherichia coli inclusion bodies. RESULTS: N- and C-Terminal His6-tagged recombinant (r) AGAP (NHis6-rAGAP and CHis6-rAGAP, respectively) were expressed in E. coli; the purification and refolding conditions were optimized. CHis6-rAGAP, but not NHis6-rAGAP, exhibited significant in vitro antihepatoma activity that was much greater than that of rAGAP produced using SUMO fusion technology (IC50, 0.4 ± 0.08 vs. 1.8 ± 0.3 µM). CHis6-rAGAP also showed significant inhibition of tumor growth in a mouse xenograft model of human hepatoma and inhibition of neuronal excitability, demonstrated by blockage of voltage-sensitive tetrodotoxin-resistant (TTX-R) sodium currents in acute isolated dorsal root ganglion neurons. CONCLUSIONS: This refolding protocol optimized for C-terminal His6-tagged scorpion rAGAP is potentially applicable to similar long-chain and cysteine-rich toxins.

Validation of AAC-11-Derived Peptide Anti-Tumor Activity in a Single Graft Sézary Patient-Derived Xenograft Mouse Model.

Sézary syndrome (SS) is an aggressive cutaneous T cell lymphoma with poor prognosis mainly characterized by the expansion of a tumor CD4+ T cell clone in both skin and blood. So far, the development of new therapeutic strategies has been hindered by a lack of reproducible in vivo models closely reflecting patients' clinical features. We developed an SS murine model consisting of the intravenous injection of Sézary patients' PBMC, together with a mixture of interleukins, in NOD-SCID-gamma mice. Thirty-four to fifty days after injection, mice showed skin disorders similar to that observed in patients, with the detection of epidermis thickening and dermal tumor T cell infiltrates. Although experimental variability was observed, Sézary cells could be tracked in the blood stream, confirming that our model could efficiently exhibit both skin and blood involvement. Using this model, we evaluated the therapeutic potential of RT39, a cell-penetrating peptide derived from the survival protein anti-apoptosis clone 11 (AAC-11), that we previously characterized as specifically inducing apoptosis of Sézary patients' malignant clone ex vivo. Systemic administration of RT39 led to cutaneous tumor T cells depletion, demonstrating efficient malignant cells' targeting and a favorable safety profile. These preclinical data confirmed that RT39 might be an innovative therapeutic tool for Sézary syndrome.

Anti-tumor peptide SA12 inhibits metastasis of MDA-MB-231 and MCF-7 breast cancer cells via increasing expression of the tumor metastasis suppressor genes, CDH1, nm23-H1 and BRMS1.

In recent years, there has been progress in the treatment of breast cancer; however, the prognosis is still poor due to recurrence and metastasis following conventional treatment. The anti-tumor peptide SA12 has been demonstrated to inhibit proliferation and arrest the cell cycle in MDA-MB-231 and MCF-7 breast cancer cells. In the present study, whether SA12 was able to inhibit the metastasis of breast cancer cells was investigated. Wound healing and Transwell assays were used to investigate the inhibition of SA12 on cell migration while, reverse transcription-quantitative PCR and western blot assays were used to identify the mechanism of action behind the effects of SA12 on cell migration. Results from the wound healing and Transwell assays revealed that SA12 significantly inhibited the migration of MDA-MB-231 and MCF-7 breast cancer cells following treatment with 100 µM SA12. Compared with that in the controls, the mRNA expression levels of cadherin 1 (CDH1), non-metastasis 23-H1 (nm23-H1) and breast cancer metastasis suppressor 1 (BRMS1) were increased in MDA-MB-231 and MCF-7 cells following treatment with 100 µM SA12. Furthermore, the protein expression levels of E-cadherin, NM23A and BRMS1 were also increased in MDA-MB-231 cells and MCF-7 cells following treatment with 100 µM SA12. In conclusion, SA12 inhibited the migration of MDA-MB-231 and MCF-7 breast cancer cells and enhanced the expression of the tumor metastasis suppressor genes, CDH1, nm23-H1 and BRMS1, which may be responsible for the SA12-induced inhibition of breast cancer cell metastasis.

Advances in effectively screening anti-tumor peptides / 药学学报

Malignant tumor is a disease that severely threaten human health. Common chemotherapeutical drugs currently used in clinical practice have some problems in severe side effects and chemoresistance. In contrast, natural venom peptides and artificially designed targeting peptides have excellent biological activities and potential druggability due to their small molecular weights and high affinity to tumor tissues. Thus, the methods for the discovery of anti-tumor peptides have attracted much attention. In this paper, we summarized the types of anti-tumor peptides from recent literatures. Then, we systematically reviewed screening theories, methods and applications based on traditional chromatographic separation, peptidomics, phage display, phenotypic screening, and artificial intelligence. These strategies and technologies will provide a methodological reference for accelerating anti-tumor peptides research.

Bio-analytical method based on MALDI-MS analysis for the quantification of CIGB-300 anti-tumor peptide in human plasma.

A fully validated bio-analytical method based on Matrix-Assisted-Laser-Desorption/Ionization-Time of Flight Mass Spectrometry was developed for quantitation in human plasma of the anti-tumor peptide CIGB-300. An analog of this peptide acetylated at the N-terminal, was used as internal standard for absolute quantitation. Acid treatment allowed efficient precipitation of plasma proteins as well as high recovery (approximately 80%) of the intact peptide. No other chromatographic step was required for sample processing before MALDI-MS analysis. Spectra were acquired in linear positive ion mode to ensure maximum sensitivity. The lower limit of quantitation was established at 0.5 µg/mL, which is equivalent to 160 fmol peptide. The calibration curve was linear from 0.5 to 7.5 µg/mL, with R(2)>0.98, and permitted quantitation of highly concentrated samples evaluated by dilution integrity testing. All parameters assessed for five validation batches met the FDA guidelines for industry. The method was successfully applied to analysis of clinical samples obtained in a phase I clinical trial following intravenous administration of CIGB-300 at a dose of 1.6 mg/kg body weight. With the exception of Cmax and AUC, pharmacokinetic parameters were similar for ELISA and MALDI-MS methods.

Combination of polypeptide AP25 and docetaxel in the treatment of breast cancer / 中国药理学通报

Aim To evaluate whether the combination of polypeptide AP25 and docetaxel is more efficient in treating experimental breast cancer,than either reagent used alone,and to offer suggestions for clinical use. Methods An experimental breast carcinoma model was set up to investigate the anti-tumor effects of AP25 and docetaxel combination.The Q value was caluculat-ed by Guinness rules and the anti-tumor effects of the combination of polypeptide AP25 and docetaxel were e-valuated.Results The treatment by the combination of polypeptide AP25 and docetaxel showed a better tumor inhibition rate.The combination of AP25 20 mg ·kg -1 and docetaxel 10 mg·kg -1 significantly inhibi-ted the tumor growth with 0.85 1.15,showing a synergistic effect.Conclusions The combination of AP25 and docetaxel can significantly in-hibit the tumor growth with a synergistic effect and de-crease the dose of chemotherapy.