Novel sequential therapy with metformin enhances the effects of cisplatin in testicular germ cell tumours via YAP1 signalling.

BACKGROUND: Testicular germ cell tumours (TGCTs) are the most commonly diagnosed malignancy in young men. Although cisplatin has been shown to be effective to treat TGCT patients, long-term follow-up has shown that TGCT survivors who accepted cisplatin treatment suffered from a greater number of adverse reactions than patients who underwent orchiectomy alone. As metformin has shown an anticancer effect in various cancers, we investigated whether metformin could enhance the effects of cisplatin to treat TGCTs. METHODS: The anticancer effects of different treatment strategies consisting of metformin and cisplatin in TCam-2 and NTERA-2 cells were assessed in vitro and in vivo. First, we used a colony formation assay, CCK-8 and MTT assays to explore the viability of TGCT cells. Flow cytometry was used to assess the cell cycle and apoptosis of TGCTs. Then, Western blotting was used to detect the protein expression of TGCTs cells after different treatments. In addition, a xenograft model was used to investigate the effects of the different treatments on the proliferation of TGCT cells. Immunohistochemistry assays were performed to analyse the expression of related proteins in the tissues from the xenograft model. RESULTS: Metformin inhibited the proliferation of TCam-2 and NTERA-2 cells by arresting them in G1 phase, while metformin did not induce apoptosis in TGCT cells. Compared with cisplatin monotherapy, the CCK-8, MTT assay and colony formation assay showed that sequential treatment with metformin and cisplatin produced enhanced anticancer effects. Further study showed that metformin blocked the cells in G1 phase by inducing phosphorylated YAP1 and reducing the expression of cyclin D1, CDK6, CDK4 and RB, which enhanced the chemosensitivity of cisplatin and activated the expression of cleaved caspase 3 in TGCTs. CONCLUSIONS: Our study discovers the important role of YAP1 in TGCTs and reports a new treatment strategy that employs the sequential administration of metformin and cisplatin, which can reduce the required cisplatin dose and enhance the sensitivity of TGCT cells to cisplatin. Therefore, this sequential treatment strategy may facilitate the development of basic and clinical research for anticancer therapies to treat TGCTs.

Low-intensity pulsed ultrasound ameliorates erectile dysfunction induced by bilateral cavernous nerve injury through enhancing Schwann cell-mediated cavernous nerve regeneration.

BACKGROUND: Cavernous nerve injury-induced erectile dysfunction caused by pelvic surgery or trauma is refractory to conventional medications and required an alternative treatment. Low-intensity pulsed ultrasound is a noninvasive mechanical therapy that promotes nerve regeneration. OBJECTIVES: To investigate the therapeutic effect and potential mechanism of low-intensity pulsed ultrasound in the treatment of neurogenic erectile dysfunction. MATERIALS AND METHODS: Thirty rats were randomly divided into the sham-operated group, bilateral cavernous nerve injury group, and bilateral cavernous nerve injury + low-intensity pulsed ultrasound group. The erectile function was assessed 3 weeks after daily low-intensity pulsed ultrasound treatment. The penile tissues and cavernous nerve tissues were harvested and subjected to histologic analysis. Primary Schwann cells and explants were extracted from adult rats. The effects of low-intensity pulsed ultrasound on proliferation, migration, and nerve growth factor expression of Schwann cells and axonal elongation were examined in vitro. RNA sequencing and western blot assay were applied to predict and verify the molecular mechanism of low-intensity pulsed ultrasound-induced Schwann cell activation. RESULTS: Our study showed that low-intensity pulsed ultrasound promoted Schwann cells proliferation, migration, and neurotrophic factor nerve growth factor expression. Meanwhile, low-intensity pulsed ultrasound exhibits a stronger ability to enhance Schwann cells-mediated neurite outgrowth of major pelvic ganglion neurons and major pelvic ganglion/cavernous nerve explants in vitro. In vivo experiments demonstrated that the erectile function of the rats in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group was significantly higher than those in the bilateral cavernous nerve injury groups. Moreover, the expression levels of smooth muscle and cavernous endothelium also increased significantly in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group. In addition, we observed the higher density and number of cavernous nerve regenerating axons in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group, indicating that low-intensity pulsed ultrasound promotes axonal regeneration following cavernous nerve injury in vivo. RNA sequencing analysis and bioinformatic analysis suggested that low-intensity pulsed ultrasound might trigger the activation of the PI3K/Akt pathway. Western blot assay confirmed that low-intensity pulsed ultrasound activated Schwann cells through TrkB/Akt/CREB signaling. CONCLUSIONS: Low-intensity pulsed ultrasound promoted nerve regeneration and ameliorated erectile function by enhancing Schwann cells proliferation, migration, and neurotrophic factor nerve growth factor expression. The TrkB/Akt/CREB axis is the possible mechanism of low-intensity pulsed ultrasound-mediated Schwann cell activation. Low-intensity pulsed ultrasound-based therapy could be a novel potential treatment strategy for cavernous nerve injury-induced neurogenic erectile dysfunction.

Preoperative systemic inflammation response index is an independent prognostic marker for BCG immunotherapy in patients with non-muscle-invasive bladder cancer.

BACKGROUND: The Systemic Inflammatory Response Index (SIRI) is a novel prognostic biomarker based on peripheral blood counts of neutrophils, monocytes, and lymphocytes. Recent evidence suggests that it is associated with poor prognosis in various cancers. However, the predictive value of the SIRI in non-muscle-invasive bladder cancer (NMIBC) patients treated with intravesical Bacillus Calmette-Guerin (BCG) immunotherapy remains elusive. Therefore, this study aimed to evaluate the potential of SIRI as a prognostic factor in these patients. METHODS: A total of 540 patients with NMIBC who underwent BCG immunotherapy following transurethral resection of bladder tumor (TURBT) were enrolled in this study. Using receiver operating characteristic (ROC) curves and the Youden index, patients were divided into high and low SIRI groups based on the cutoff values. Univariable and multivariable logistic regression analyses were performed to identify independent predictors of BCG non-response. Thereafter, propensity score matching (PSM) was used to eliminate bias due to confounding factors between the low and high SIRI groups. Finally, the Kaplan-Meier method was used to compare recurrence-free survival (RFS) and progression-free survival (PFS) between the two groups. RESULTS: Multivariable logistic regression analysis revealed that high SIRI (p = 0.001), high MLR (p = 0.015), and high tumor pathological T stage (p = 0.015) were significantly correlated with non-response to BCG therapy. In addition, both RFS and PFS were shorter in the high SIRI group than in the other group before and after PSM (both p < 0.05). Collectively, our results indicate that the combination of tumor pathological T staging and the SIRI can enhance the predictive power of BCG response. CONCLUSION: Pretreatment peripheral blood SIRI can be employed to predict the response to BCG immunotherapy and the prognosis of NMIBC patients. Taken together, the combination of T stage and SIRI demonstrated robust performance in predicting the response to BCG immunotherapy in NMIBC patients.

LIPUS-SCs-Exo promotes peripheral nerve regeneration in cavernous nerve crush injury-induced ED rats via PI3K/Akt/FoxO signaling pathway.

OBJECTIVE: Clinical treatment of erectile dysfunction (ED) caused by cavernous nerve (CN) injury during pelvic surgery is difficult. Low-intensity pulsed ultrasound (LIPUS) can be a potential strategy for neurogenic ED (NED). However, whether Schwann cells (SCs) can respond to LIPUS stimulation signals is unclear. This study aims to elucidate the signal transmission between SCs paracrine exosome (Exo) and neurons stimulated by LIPUS, as well as to analyze the role and potential mechanisms of exosomes in CN repair after injury. METHODS: The major pelvic ganglion (MPG) neurons and MPG/CN explants were stimulated with LIPUS of different energy intensities to explore the appropriate LIPUS energy intensity. The exosomes were isolated and purified from LIPUS-stimulated SCs (LIPUS-SCs-Exo) and non-stimulated SCs (SCs-Exo). The effects of LIPUS-SCs-Exo on neurite outgrowth, erectile function, and cavernous penis histology were identified in bilateral cavernous nerve crush injury (BCNI)-induced ED rats. RESULTS: LIPUS-SCs-Exo group can enhance the axon elongation of MPG/CN and MPG neurons compared to SCs-Exo group in vitro. Then, the LIPUS-SCs-Exo group showed a stronger ability to promote the injured CN regeneration and SCs proliferation compared to the SCs-Exo group in vivo. Furthermore, the LIPUS-SCs-Exo group increased the Max intracavernous pressure (ICP)/mean arterial pressure (MAP), lumen to parenchyma and smooth muscle to collagen ratios compared to the SCs-Exo group in vivo. Additionally, high-throughput sequencing combined with bioinformatics analysis revealed the differential expression of 1689 miRNAs between the SCs-Exo group and the LIPUS-SCs-Exo group. After LIPUS-SCs-Exo treatment, the phosphorylated levels of Phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and forkhead box O (FoxO) in MPG neurons increased significantly compared to negative control (NC) and SCs-Exo groups. CONCLUSION: Our study revealed that LIPUS stimulation could regulate the gene of MPG neurons by changing miRNAs derived from SCs-Exo, then activating the PI3K-Akt-FoxO signal pathway to enhance nerve regeneration and restore erectile function. This study had important theoretical and practical significance for improving the NED treatment.

Intracavernous Pressure Recording in a Cavernous Nerve Injury Rat Model.

The bilateral cavernous nerve (CN) injury rat model has been extensively used to simulate clinical cavernous nerve injury associated with erectile dysfunction (ED) for evaluating the effect of clinical therapeutic methods. However, the methods of CN injury model construction are flawed and varied in the ED research field. It is CN crush injury that is the most commonly used method in recent years. This study aims to provide a detailed description of the procedure of bilateral CN injury rat model construction and measurement of intracavernous pressure (ICP) recording, providing a reliable and reproducible CN injury rat model. This work successfully developed the CN injury method of hemostat crush injury using a syringe needle as hard support and a hemostat with a rubber sleeve. Also, this method concludes that a voltage of 1.0 V, frequency of 20 Hz, and pulse-width of 5 ms are the optimized stimulation parameters for ICP recording in a bilateral CN injury rat model.

Synthesis of a novel platinum(II) complex with 6,7-dichloro-5,8-quinolinedione and the study of its antitumor mechanism in testicular seminoma.

A new platinum(II) complex, [Pt(ClClQ)(DMSO)Cl] (1), utilizing 6,7-dichloro-5,8-quinolinedione (ClClQ) as a ligand, has been synthesized and fully characterized. Single-crystal X-ray diffraction and other spectroscopic and analytical methods revealed that the coordination geometry of Pt(II) in complex 1 can also be described as a four-coordinated square planar geometry. The aim of the study was to explore the in vitro anticancer properties of complex 1. Our studies showed that complex 1 can regulate the viability of testicular seminoma cells in vitro, including cell proliferation and apoptosis. We further observed negative regulation by complex 1 of the expression levels of the key elements in the phosphoinositide-3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK3ß) pathway, including phosphorylated phosphoinositide-3 kinase (p-PI3K), phosphorylated protein kinase B(p-Akt) and phosphorylated glycogen synthase kinase-3ß (p-GSK3ß). Moreover, the negative effect of complex 1 was reversed by LiCl, a GSK3ß-specific inhibitor of the PI3K signaling pathway. Meanwhile, the levels of Bcl2 associated death promoter (Bad), cytochrome c, active-caspase-3 and active-caspase-9 increased significantly. In conclusion, we observed that complex 1 can regulate the viability of testicular seminoma cells through the PI3K/Akt/GSK3ß signaling pathway and the mitochondria-mediated apoptotic pathway in vitro, and thus, complex 1 may have potential for use as a drug in the treatment of testicular germ cell tumors.