KLF2 controls proliferation and apoptosis of human spermatogonial stem cells via targeting GJA1.

Human spermatogonial stem cells (SSCs) are essential for spermatogenesis and male fertility. However, molecular mechanisms regulating fate determinations of human SSCs remain elusive. In this study, we revealed that KLF2 decreased the proliferation, DNA synthesis, and colonization of human SSCs as well as increased apoptosis of these cells. We identified and demonstrated that GJA1 was a target gene for KLF2 in human SSCs. Notably, KLF2 overexpression rescued the reduction of proliferation of human SSCs caused by GJA1 silencing as well as the enhancement of apoptosis of human SSCs. Abnormalities in the higher level of KLF2 and/or KIF2 mutations might lead to male infertility. Collectively, these results implicate that KLF2 inhibits proliferation of human SSCs and enhances their apoptosis by targeting GJA1. This study thus provides novel genetic mechanisms underlying human spermatogenesis and azoospermia, and it offers new endogenous targets for treating male infertility.

WNT5A regulates the proliferation, apoptosis and stemness of human stem Leydig cells via the ß-catenin signaling pathway.

Stem Leydig cells (SLCs) are essential for maintaining normal spermatogenesis as the significant component of testis microenvironment and gonadal aging. Although progress has been achieved in the regulation of male germ cells in mammals and humans, it remains unknown about the genes and signaling pathways of human SLCs. Here we have demonstrated, for the first time, that WNT5A (Wnt family member 5a) mediates the proliferation, apoptosis, and stemness of human SLCs, namely NGFR+ Leydig cells. We revealed that NGFR+ Leydig cells expressed NGFR, PDGFRA, NES, NR2F2, and THY1, hallmarks for SLCs. RNA-sequencing showed that WNT5A was expressed at a higher level in human SLCs than non-SLCs, while immunohistochemistry and Western blots further illustrated that WNT5A was predominantly expressed in human SLCs. Notably, CCK-8, EdU and Western blots displayed that WNT5A enhanced the proliferation and DNA synthesis and retained stemness of human SLCs, whereas flow cytometry and TUNEL analyses demonstrated that WNT5A inhibited the apoptosis of these cells. WNT5A knockdown caused an increase in LC lineage differentiation of human SLCs and reversed the effect of WNT5A overexpression on fate decisions of human SLCs. In addition, WNT5A silencing  resulted in the decreases in nuclear translocation of ß-catenin and expression levels of c-Myc, CD44, and Cyclin D1. Collectively, these results implicate that WNT5A regulates the proliferation, apoptosis and stemness of human SLCs through the activation of the ß-catenin signaling pathway. This study thus provides a novel molecular mechanism underlying the fate determinations of human SLCs, and it offers a new insight into the niche regulation of human testis.

The beneficial effects of alpha-tocopherol on intestinal function and the expression of tight junction proteins in differentiated segments of the intestine in piglets.

Alpha (α)-tocopherol is a major component of dietary vitamin E. Despite being one of the most widely used food supplements in both animals and humans, its role in intestinal functions remains unknown. We were able to examine and accurately demonstrate its permeability effect in vitro and its differentiated effect on tight junction expression in different segments of the intestine in vivo using cultured intestinal porcine epithelial cell line (IPEC-J2) and piglets. A cultured IPEC-J2 demonstrated that α-tocopherol upregulated the expression of tight junction proteins and improved their integrity, with a maximum effect at concentrations ranging from 20 to 40 µmol/L. In vivo data from weaned pigs fed different doses of α-tocopherol for 2 weeks revealed that α-tocopherol effectively increases the expression of tight junction proteins in all sections of the intestinal mucosa, with the highest effect on the duodenum at an optimum dose of 20-50 mg/kg. In contrast, α-tocopherol did not affect intestinal inflammation. These findings suggest that α-tocopherol maintains intestinal integrity and increases the expression of tight junction proteins both in vitro and in vivo.

Vitamin A prevents lipopolysaccharide-induced injury on tight junctions in mice.

Vitamin A (VA) is one of the most widely used food supplements, but its molecular mechanisms largely remain elusive. Previously, we have demonstrated that VA inhibits the action of lipopolysaccharide (LPS) on intestinal epithelial barrier function and tight junction proteins using IPEC-J2 cells, one of representative intestinal cell lines as a cellular model. These exciting findings stimulated us continue to determine the effects of VA on LPS-induced damage of intestinal integrity in mice. Our results demonstrated that LPS treatment caused reductions of the mRNA levels of tight junction proteins including Zo-1, Occludin, and Claudin-1, well-known biomarkers of intestinal integrity, and these reductions were reversed by VA pretreatment. Intestinal immunofluorescent results of Claudin-1 revealed that LPS disrupted the structure of tight junction and reduced the expression of Claudin-1 at protein level, which was reversed by VA pretreatment. These results suggest that VA may exert a profound role on preventing intestinal inflammation in vivo.

Betaine attenuates LPS-induced downregulation of Occludin and Claudin-1 and restores intestinal barrier function.

BACKGROUND: The intestinal epithelial barrier, which works as the first line of defense between the luminal environment and the host, once destroyed, it will cause serious inflammation or other intestinal diseases. Tight junctions (TJs) play a vital role to maintain the integrity of the epithelial barrier. Lipopolysaccharide (LPS), one of the most important inflammatory factors will downregulate specific TJ proteins including Occludin and Claudin-1 and impair integrity of the epithelial barrier. Betaine has excellent anti-inflammatory activity but whether betaine has any effect on TJ proteins, particularly on LPS-induced dysfunction of epithelial barriers remains unknown. The purpose of this study is to explore the pharmacological effect of betaine on improving intestinal barrier function represented by TJ proteins. Intestinal porcine epithelial cells (IPEC-J2) were used as an in vitro model. RESULTS: The results demonstrated that betaine enhanced the expression of TJ proteins while LPS (1 µg/mL) downregulates the expression of these proteins. Furthermore, betaine attenuates LPS-induced decreases of TJ proteins both shown by Western blot (WB) and Reverse transcription-polymerase chain reaction (RT-PCR). The immunofluorescent images consistently revealed that LPS induced the disruption of TJ protein Claudin-1 and reduced its expression while betaine could reverse these alterations. Similar protective role of betaine on intestinal barrier function was observed by transepithelial electrical resistance (TEER) approach. CONCLUSION: In conclusion, our research demonstrated that betaine attenuated LPS-induced downregulation of Occludin and Claudin-1 and restored the intestinal barrier function.

Ultrasensitive detection of microRNA-21 based on electrophoresis assisted cascade chemiluminescence signal amplification for the identification of cancer cells.

Rapid and accurate detection of microRNA content in cells is of great significance. Here, an ultrasensitive microchip electrophoresis (MCE) method based on cascade chemiluminescence (CL) signal amplification was developed for the detection of microRNA-21 in cells. In this method, horseradish peroxidase labeled DNA was used as a signal probe, which could induce CL signal by the reaction of luminol and H2O2. Combining with two cyclic enzyme digestion reactions by T7 exonuclease, a large number of signal probes were degraded. By using MCE-CL as a separation and detection platform, an amplified CL signal peak was achieved. The developed MCE-CL method can detect miR-21 at a concentration as low as 1.0 × 10-15 M, which was enhanced by six orders of magnitude compared with those of conventional MCE-CL assay. This method has been applied for the detection of microRNA-21 in cell lysate, which show that there were significant differences of miR-21 among different types of cells, and the content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, the proposed method held great application potential in early diagnosis of tumor and biomedical research.

Synthesis, biological evaluation and anti-proliferative mechanism of fluorine-containing proguanil derivatives.

Proguanil, a member of biguanide family, has excellent anti-proliferative activities. Fluorine-containing compounds have been demonstrated to have super biological activities including enhanced binding interactions, metabolic stability, and reduced toxicity. In this study, based on the intermediate derivatization methods, we synthesized 13 new fluorine-containing proguanil derivatives, and found that 7a,7d and 8e had much lower IC50 than proguanil in 5 human cancerous cell lines. The results of clonogenic and scratch wound healing assays revealed that the inhibitory effects of derivatives 7a,7d and 8e on proliferation and migration of human cancer cell lines were much better than proguanil as well. Mechanistic study based on representative derivative 7a indicated that this compound up-regulates AMPK signal pathway and downregulates mTOR/4EBP1/p70S6K. In conclusion, these new fluorine-containing derivatives show potential for the development of cancer chemotherapeutic drugs.

Dual Inhibition of Pirarubicin-Induced AKT and ERK Activations by Phenformin Sensitively Suppresses Bladder Cancer Growth.

Activations of Akt or ERK pathway induced by clinical drugs promote therapeutic failure due to decrease of drug response, and no available strategies have been developed to solve these problems. In this study, we found that pirarubicin (THP), one important chemotherapeutic drug for treating bladder cancer intravesically, dramatically elevated phosphorylations of both Akt and Erk1/2 in addition to inducing DNA damage. MK2206 or AZD6244, representative Akt and Erk1/2 inhibitors, respectively, profoundly sensitized bladder cancer cells to THP treatment. Interestingly, we found that inhibition of a single arm of either Akt or Erk1/2 pathway would induce the increase of another arm, indicating the existence of the crosstalk between these two pathways. Thus, simultaneous suppression of both signals may be needed for increasing the sensitivity of THP. On the other hand, we revealed that phenformin efficiently inhibited both Akt and Erk1/2 phosphorylation in a dose-dependent manner. Furthermore, we demonstrated that phenformin, mimicking dual inhibitors, plays dramatically synergistic action with THP both in vitro and in vivo. Our findings suggest that combination therapy of THP with dual inhibitors may constitute a successful strategy for improving chemotherapy response.

Anticancer properties of novel pyrazole-containing biguanide derivatives with activating the adenosine monophosphate-activated protein kinase signaling pathway.

Biguanides, including metformin and phenformin, have emerged as promising anticancer agents. However, the high dose needed for their efficient anticancer properties restricts their clinical application. In an attempt to obtain higher active compounds than these parent compounds, pyrazole-containing biguanide derivatives were synthesized and screened for in vitro cytotoxicity against human cancer cell lines. Clonogenic assays and scratch wound healing assays demonstrated that these new derivatives profoundly inhibit cell proliferation and migration. Compounds 10b and 10d exhibited strong potency with low IC50 values in the range of 6.9-28.3 µM, far superior to phenformin and metformin. Moreover, 20 µM 10b and 10d resulted in 72.3-88.2% (p < 0.001) inhibition of colony formation and 29.3-60.7% (p < 0.05) inhibition of cell migration. Mechanistically, 10b and 10d activated adenosine monophosphate-activated protein kinase, leading to inactivation of the mammalian target of rapamycin (mTOR) signaling pathway with the regulation of 4EBP1 and p70S6K. These results suggest the value of these novel biguanide derivatives as candidates with therapeutic potential for the treatment of bladder and ovarian cancer.

Vitamin A inhibits the action of LPS on the intestinal epithelial barrier function and tight junction proteins.

Inflammation caused by either intrinsic or extrinsic toxins results in intestinal barrier dysfunction, contributing to inflammatory bowel disease (IBD) and other diseases. Vitamin A is a widely used food supplement although its mechanistic effect on intestinal structures is largely unknown. The goal of this study was to explore the mechanism by investigating the influence of vitamin A on the intestinal barrier function, represented by tight junctions. IPEC-J2 cells were differentiated on transwell inserts and used as a model of intestinal barrier permeability. Transepithelial electrical resistance (TEER) was used as an indicator of monolayer integrity and paracellular permeability. Western blot and the reverse transcriptase-polymerase chain reaction were used to assess the protein and mRNA expression of tight junction proteins. Immunofluorescence microscopy was used to evaluate the localization and expression of tight junctions. Differentiated cells were treated with a vehicle control (Ctrl), inflammatory stimulus (1 µg mL-1 LPS), LPS co-treatment with 0.1 µmol L-1 Vitamin A (1 µg mL-1 LPS + 0.1 µmol L-1 VA) and 0.1 µmol L-1 Vitamin A. LPS significantly decreased TEER by 24 hours, continuing this effect to 48 hours after application. Vitamin A alleviated the LPS-induced decrease of TEER from 12 hours to 48 hours, while Vitamin A alone enhanced TEER, indicating that Vitamin A attenuated LPS-induced intestinal epithelium permeability. Mechanistically, different concentrations of Vitamin A (0-20 µmol L-1) enhanced tight junction protein markers including Zo-1, Occludin and Claudin-1 both at protein and mRNA levels with an optimized dose of 0.1 µmol L-1. Immunofluorescence results demonstrated that majority of Zo-1 and Claudin-1 is located at the tight junctions, as we expected. LPS reduced the expression of these proteins and Vitamin A reversed LPS-reduced expression of these proteins, consistent with the results of western blot. In conclusion, Vitamin A improves the intestinal barrier function and reverses LPS-induced intestinal barrier damage via enhancing the expression of tight junction proteins.

Down-regulation of PKM2 decreases FASN expression in bladder cancer cells through AKT/mTOR/SREBP-1c axis.

Fatty acid synthase (FASN) catalyzing the terminal steps in the de novo biogenesis of fatty acids is correlated with low survival and high disease recurrence in patients with bladder cancer. Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels and provides a growth advantage to tumors. However, it is unclear whether the change of PKM2 has an effect on FASN and what is the mechanisms underlying. Here we describe a novel function of PKM2 in control of lipid metabolism by mediating transcriptional activation of FASN, showing the reduced expression of sterol regulatory element binding protein 1c (SREBP-1c). We first discovered that PKM2 physically interacts with the SREBP-1c using biochemical approaches, and downregulation of PKM2 reduced the expression of SREBP-1c by inactivating the AKT/mTOR signaling pathway, which in turn directly suppressed the transcription of major lipogenic genes FASN to reduce tumor growths. Furthermore, either PKM2 inhibitor-Shikonin or FASN inhibitor-TVB-3166 alone induced a strong antiproliferative and anticolony forming effect in bladder cancer cell line. The combination of both inhibitors exhibits a super synergistic effect on blocking the bladder cancer cells growth. It provides a new target and scientific basis for the treatment of bladder cancer.

Phenformin alone or combined with gefitinib inhibits bladder cancer via AMPK and EGFR pathways.

BACKGROUND: In previous studies, we have shown that the combination of metformin and gefitinib inhibits the growth of bladder cancer cells. Here we examined whether the metformin analogue phenformin, either used alone or in combination with gefitinib, could inhibit growth of bladder cancer cells. METHODS: The growth-inhibitory effects of phenformin and gefitinib were tested in one murine and two human bladder cancer cell lines using MTT and clonogenic assays. Effects on cell migration were assessed in a wound healing assay. Synergistic action between the two drugs was assessed using CompuSyn software. The potential involvement of AMPK and EGFR pathways in the effects of phenformin and gefitinib was explored using Western blotting. RESULTS: In MTT and clonogenic assays, phenformin was > 10-fold more potent than metformin in inhibiting bladder cancer cell growth. Phenformin also potently inhibited cell migration in wound healing assays, and promoted apoptosis. AMPK signaling was activated; EGFR signaling was inhibited. Phenformin was synergistic with gefitinib, with the combination of drugs showing much stronger anticancer activity and apoptotic activation than phenformin alone. CONCLUSIONS: Phenformin shows potential as an effective drug against bladder cancer, either alone or in combination with gefitinib.

One-pot synthesis of a metal-organic framework-based drug carrier for intelligent glucose-responsive insulin delivery.

We have developed a glucose-responsive metal-organic framework (MOF)-based insulin delivery nanosystem via a one-pot process. The system relies on the MOF response to glucose stimulation and this can promote insulin delivery. This nanosystem was successfully applied for glucose-responsive and self-regulated insulin release.

A novel synthetic derivative of quercetin, 8-trifluoromethyl-3,5,7,3',4'-O-pentamethyl-quercetin, inhibits bladder cancer growth by targeting the AMPK/mTOR signaling pathway.

Quercetin is a naturally existing compound and shows attractive anticancer properties for a variety of solid tumors including glioma, bladder cancer, hepatocellular carcinoma, breast cancer, hematological malignancies and prostate carcinoma. However, these anticancer properties have not been clinically approved due to unclear mechanistic information and its low bioactivity. In our previous study, we elucidated that quercetin activates AMPK pathway which is the major mechanism for its unique anticancer effect in bladder cancer. In the present study, we are trying to enhance its bioactivity by chemical modification using fluorination approach to prepare novel chemical entities, based on the principle of intermediate derivative method (IDM). The compound we obtained is named 8-trifluoromethyl-3,5,7,3',4'-O-pentamethyl- quercetin (TFQ), characterized by NMR spectra and mass spectrum (MS). The results from MTT and cologenic assay in two human and one murine bladder cancer cell lines showed that TFQ exhibits more potent inhibition on the three bladder cancer cell lines than quercetin (Que) although this enhanced effects is not very dramatic. Furthermore, we found that the survival of normal bladder cells PEBC was not significantly suppressed by TFQ compared with Que. Western blot analysis showed that TFQ possess more potent AMPK activation than Que. The downstream of AMPK was further examined by western blot. TFQ treatment is able to inactivate mTOR signaling pathway with the regulation of mTOR, 4EBP1 and P70S6K. These results demonstrated that the fluorinated quercetin derivative TFQ inhibits bladder cancer cell growth through the AMPK/mTOR pathway. Altogether, our findings suggest that TFQ could serve as a new potential therapeutic agent for bladder cancer more effective than Que.

Human interstitial cellular model in therapeutics of heart valve calcification.

Calcific aortic valve disease is a common, severe heart condition that is currently with no proven, effective drug treatment and requires a surgical valve replacement or an entire heart explanation. Thus, developing novel, targeted therapeutic approaches becomes a major goal for cardiovascular disease research. To achieve this goal, isolated heart valve interstitial cells could be an advanced model to explore molecular mechanisms and measure drug efficacy. Based on this progress, molecular mechanisms that harbor components of  inflammation and fibrosis coupled with proteins, for example, BMP-2, TLRs, RANKL, Osteoprotegerin, have been proposed. Small molecules or antibodies targeting these proteins have shown promising efficacy for either reversing or slowing down calcification development in vitro. In this review, we summarize these potential therapeutics with some highlights of interstitial cellular models.

Resistant Starch Regulates Gut Microbiota: Structure, Biochemistry and Cell Signalling.

Starch is one of the most popular nutritional sources for both human and animals. Due to the variation of its nutritional traits and biochemical specificities, starch has been classified into rapidly digestible, slowly digestible and resistant starch. Resistant starch has its own unique chemical structure, and various forms of resistant starch are commercially available. It has been found being a multiple-functional regulator for treating metabolic dysfunction. Different functions of resistant starch such as modulation of the gut microbiota, gut peptides, circulating growth factors, circulating inflammatory mediators have been characterized by animal studies and clinical trials. In this mini-review, recent remarkable progress in resistant starch on gut microbiota, particularly the effect of structure, biochemistry and cell signaling on nutrition has been summarized, with highlights on its regulatory effect on gut microbiota.

Combination of metformin with chemotherapeutic drugs via different molecular mechanisms.

Metformin, a widely prescribed drug for treating type II diabetes, is one of the most extensively recognized metabolic modulators which has shown an important anti-cancer property. However, fairly amount of clinical trials on its single administration have not demonstrated a convincing efficiency yet. Thus, recent studies tend to combine metformin with clinical commonly used chemotherapeutic drugs to decrease their toxicity and attenuate their tumor resistance. These strategies have displayed promising clinical benefits. Interestingly, metformin experiences a diversity of molecular mechanisms when it combines different chemotherapeutic drugs. For example, AMPK/mTOR signaling pathway activation plays a major role when it combines with hormone modulating drugs. In contrast, suppression of HIF-1, p-gp and MRP1 protein expression is its main mechanism when metformin combines with anti-metabolites. Furthermore, when combining of metformin with antibiotics, inhibition of oxidative stress and inflammatory signaling pathway becomes a novel pharmaceutical mechanism for its cardio-protective effect. Induction of apoptotic mitochondria and nucleus could be the major player for the synergistic effect of its combination with cisplatin. In contrast, down-regulation of lipoprotein or cholesterol synthesis might be the undefined molecular base when metformin combines with taxane. Thus, deep exploration of molecular mechanisms of metformin with these different drugs is critical to understand its synergistic effect and help for personalized administration. In this mini-review, detailed molecular mechanisms of these combinations are discussed and summarized. This work will promote better understanding of molecular mechanisms of metformin and provide precise targets to identify specific patient groups to achieve satisfactory treatment efficacy.