Minnelide Markedly Reduces Proteinuria in Mice with Adriamycin Nephropathy by Protecting Against Podocyte Injury.

Minimal change disease (MCD) is the most common cause of idiopathic nephrotic syndrome in children. The current major therapy is hormones for most steroid-sensitive patients. However, many patients have recurrent relapses of the disease and require long-term immunosuppression, leading to significant morbidity due to the side effects of the drugs. Therefore, better drugs need to be urgently explored to treat nephrotic syndrome while avoiding the side effects of drugs. Minnelide, a water-soluble prodrug of triptolide, has been proved to be effective in treating cancers in many clinical trials. This study aimed to investigate the therapeutic effect of minnelide in mice with adriamycin (ADR) nephropathy, its underlying protection mechanisms, and its reproductive toxicity. Minnelide was administered intraperitoneally to 6-8-week female mice with adriamycin nephropathy for 2 weeks, and the urine, blood, and kidney tissues were taken to analyze the therapeutic effect. In addition, we evaluated reproductive toxicity by measuring the levels of gonadal hormones and observing the histological changes in ovaries and testes. Primary mouse podocytes were exposed to puromycin (PAN) to damage the cytoskeleton and induce apoptosis, and then, triptolide was used to evaluate the therapeutic effect and underlying protection mechanisms in vitro. It was observed that minnelide dramatically alleviated proteinuria and apoptosis in mice with adriamycin nephropathy. In vitro, triptolide ameliorated puromycin-induced cytoskeletal rearrangement and apoptosis via reactive oxygen species-mediated mitochondrial pathway. In addition, minnelide caused no reproductive toxicity to male and female mice. The results suggested that minnelide might be a promising drug for nephrotic syndrome.

Conjugation of Cetuximab - Puromycin and Its Target-Specific Effect on Triple Negative Breast Cancer Cell Lines.

Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also toxic for normal cell, not specific to the target cells. Consequently, chemotherapy has various side effects. Monoclonal antibody (MAb) has been developed for specific therapy which only has killing effect in cancer cells, but the survival rate of most MAbs around 20%. Therefore, in clinical practice, MAbs administration should combine with chemotherapeutic agents. For effectiveness of therapy and to minimalize adverse effects, anticancer agent with selective cytotoxic effect on target cells is needed, the immunotoxin. OBJECTIVE: This study introduces a novel approach to conjugate monoclonal antibody (Cetuximab) and toxin (Puromycin), in order to selectively inhibit proliferation of triple negative breast cancer (TNBC) and to enhance the efficacy of MAb in target cells killing. METHODS: Cetuximab was conjugated with Puromycin using a linker, i.e SATP (Succinimidyl-acetylthiopropionate) and tested on triple negative breast cancer cell lines (MDA-MB-231) which expressed EGFR (epidermal growth factor receptor). Cetuximab is MAb which targets EGFR. MCF-7 was used as control cells since it has low or no EGFR expression. Cell counting were conducted as viability assay at 24 hours, 48 hours, and 72 hours after treatment. RESULTS: The results showed significant reduction of live cells number in Conjugate 20 µg/mL cultured in MDA-MB-231 compared to MCF-7 after 24 hours, 48 hours, and 72 hours incubation. In all time period of incubation, significant reduction of MDA-MB-231 live cells number was also observed in Conjugate 20 µg/mL compared to Cetuximab 20 µg/mL. CONCLUSION: Synthesized conjugate showed its target-specific effect in TNBC and improved the efficacy of Cetuximab on TNBC. In the future, this conjugate can be a potential anticancer therapy in treating triple-negative breast cancer.

Role of network-mediated stochasticity in mammalian drug resistance.

A major challenge in biology is that genetically identical cells in the same environment can display gene expression stochasticity (noise), which contributes to bet-hedging, drug tolerance, and cell-fate switching. The magnitude and timescales of stochastic fluctuations can depend on the gene regulatory network. Currently, it is unclear how gene expression noise of specific networks impacts the evolution of drug resistance in mammalian cells. Answering this question requires adjusting network noise independently from mean expression. Here, we develop positive and negative feedback-based synthetic gene circuits to decouple noise from the mean for Puromycin resistance gene expression in Chinese Hamster Ovary cells. In low Puromycin concentrations, the high-noise, positive-feedback network delays long-term adaptation, whereas it facilitates adaptation under high Puromycin concentration. Accordingly, the low-noise, negative-feedback circuit can maintain resistance by acquiring mutations while the positive-feedback circuit remains mutation-free and regains drug sensitivity. These findings may have profound implications for chemotherapeutic inefficiency and cancer relapse.

Selective cancer targeting with prodrugs activated by histone deacetylases and a tumour-associated protease.

Eradication of cancer cells while minimizing damage to healthy cells is a primary goal of cancer therapy. Highly selective drugs are urgently needed. Here we demonstrate a new prodrug strategy for selective cancer therapy that utilizes increased histone deacetylase (HDAC) and tumour-associated protease activities produced in malignant cancer cells. By coupling an acetylated lysine group to puromycin, a masked cytotoxic agent is created, which is serially activated by HDAC and an endogenous protease cathepsin L (CTSL) that remove the acetyl group first and then the unacetylated lysine group liberating puromycin. The agent selectively kills human cancer cell lines with high HDAC and CTSL activities. In vivo studies confirm tumour growth inhibition in prodrug-treated mice bearing human cancer xenografts. This cancer-selective cleavage of the masking group is a promising strategy for the next generation of anticancer drug development that could be applied to many other cytotoxic agents.

Pregnancy following chemical activation of oocytes in a couple with repeated failure of fertilization using ICSI: case report.

We report our attempts to achieve a successful pregnancy outcome with calcium ionophore A23187 and puromycin oocyte activation using sperm from a normozoospermic husband of a patient with previous repeated failed fertilization following ICSI. Oocytes from the female partner of a couple with a 4 year history of unexplained primary infertility with repeated failed fertilization following ICSI were used. In the latest ICSI attempt, oocytes were activated by treatment with calcium ionophore (5 min) and puromycin (5 h), then cultured. In this cycle, assisted oocyte activation with calcium ionophore and puromycin after ICSI resulted in a satisfactory fertilization rate (8/12; 66.7%); in prior cycles only one of 71 oocytes (1.4%) was fertilized. The outcome was a Caesarean section delivery of a healthy male infant without congenital abnormalities at 41 weeks, 2 days of gestation. In conclusion, the use of calcium ionophore and puromycin for oocyte activation was found to be a useful method in a case of repeated failed fertilization after ICSI.

Development of cardiac hypertrophy in rats exposed to acute hypobaric hypoxia. - Studies with protein synthesis inhibitors.

The study is based on the experimental model of myocardial hypertrophy in rats. Male Sprague-Dawley adult rats were exposed to acute hypoxia in a hypobaric chamber (0.40 atmospheres of air for 24 hours). Myocardial hypertrophy was detected by wet and dry heart weight values when calculated on the basis of total body weight; i.e., ratio heart weight in mg to body weight in 100 g. The behaviour of myocardial hypertrophy was assessed after treatment of intact animals with Puromycin (10 mg/rat i.p.) or Actinomycin-D (100 micrograms/rat i.p.). The experimental results showed the following dry heart weight values (mean +/- SEM): room pressure 60.5 +/- 1.5; hypoxia 69.2 +/- 0.9; Actinomycin-D + hypoxia 63.5 +/- 0.6; Puromycin + hypoxia 62.4 +/- 1.5, (P less than 0.001). From the comparison of data reveals that inhibitors of protein synthesis completely inhibit the myocardial hypertrophy due to hypoxia. Thus it is documented that acute hypobaric hypoxia is able to induce in the rat an actual myocardial hypertrophy condition as expressed of the enhanced protein synthesis.

[Effect of inhibitors of RNA and protein synthesis on audiogenic epileptiform reactions in rats of the KM line]. / Vliianie ingibitorov sinteza RNK i belkov na audiogennye épileptiformnye reaktsii u krys linii KM

Injection of actinomycin D, puromycin and cycloheximide change the genetically determined reaction of rats to acoustic stimuli up to the appearance of resistance to sound. The change in the nature of audiogenic epileptiform seizures correlates with the disturbance of the RNA and protein synthesis in the experimental animals brain. The appearance of sound resistance does not correlate with the degree of inhibition of protein synthesis in the brain. It is assumed that the antiseizure effect of the drugs used is possibly due to their action on the neuromediator systems of the brain.

[Effect of puromycin and cycloheximide on the course of audiogenic epileptic seizures in rats]. / Vliianie puromitsina i tsiklogeksimida na techenie audiogennykh épilepticheskikh pripadkov u krys

Puromycin was injected intraventricularly and cyclohexymid subcutaneously into rats with a hereditary predisposition to audiogenic epileptiform seizures. The injections inhibited the intensity of protein synthesis in the brain by 40 to 65%. Resistance to acoustic stimuli which appeared in 24% of the experimental animals did not correlate with the degree of protein synthesis inhibition in the brain.

Kinetics of multinucleate giant cell formation and their modification by various agents in foreign body reactions.

Foreign body multinucleate giant cells were produced by the implantation of a strip of Melenex in the subcutaneous tissues of mice. The implants were removed at various intervals, and the proportion of multinucleate giant cells as well as the number of nuclei they contained were counted and statistically assessed. The greatest proportion of giant cells was reached 4 weeks after implantation, when 25% of the attached cells were multinucleated. The mean nuclear content however was greatest approximately 2 weeks after implantation and rapidly fell over the ensuing weeks. The fusion potential however, remained almost unaltered for the remainder of the experimental period. Transplantation of 7-day Melenex implants from normal donors into lethally irradiated recipients demonstrated that the halflife of the giant cells is only a few days. Treatment with carrageenin, species-specific antisera, actinomycin D and cortisone inhibited, while puromycin enhanced, multinucleate cell formation. Calcium gluconate, EDTA and irradiation had no significant effect. The possible interpretation of some of these findings is discussed.