TRPC3 promotes tumorigenesis of gastric cancer via the CNB2/GSK3ß/NFATc2 signaling pathway.

The transient receptor potential canonical (TRPC) channels have been implicated in various types of malignancies including gastric cancer (GC). However, the detailed mechanisms of TRPC channels underlying cell proliferation and apoptosis of GC cells remain largely unknown. Here, we report that TRPC3 was highly expressed in clinical GC specimens and correlated with GC malignant progression and poor prognosis. Forced expression of TRPC3 in GC cells enhanced both receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) and promoted the nuclear factor of activated T cell 2 (NFATc2) nuclear translocation by AKT/GSK-3ß and CNB2 signaling. Pharmacological inhibition of TRPC3 or CRISPR/Cas9-mediated TRPC3 knockout effectively inhibited the growth of GC cells both in vitro and in vivo. These effects were reversible by the rescue of TRPC3 expression. Furthermore, we confirmed the role of TRPC3 and the ROCE-AKT/GSK3ß-CNB2/NFATc2 signaling cascade in regulating cell cycle checkpoint, apoptosis cascade, and intracellular ROS production in GC. Overall, our findings suggest an oncogenic role of TRPC3 in GC and may highlight a potential target of TRPC3 for therapeutic intervention of GC and its malignant progression.

Differential clinical benefits of continuous blood purification treatment in critically ill patients with variable APACHE II scores.

The present study aimed to assess whether the Acute Physiology And Chronic Health Evaluation (APACHE) II score may be used to predict whether critically ill patients benefit from continuous blood purification (CBP) treatment. A total of 115 critically ill patients were retrospectively reviewed and grouped according to their baseline APACHE II scores. Each group was further divided into 2 groups based on whether they received CBP or not. At 72 h after CBP treatment, clinical indicators comprising the plasma levels of inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8, as well as endotoxin and procalcitonin (PCT), and severity scores (APACHE II, multiple organ dysfunction syndrome and systemic inflammatory response syndrome), were analyzed in all patients. It was observed that while CBP slightly reduced the severity scores in all patients, it significantly improved those in patients with an APACHE II score of 20-29 (P<0.05). Similarly, the plasma levels of TNF-α, IL-6, IL-8, endotoxin and PCT were significantly lower in patients receiving CBP than in those without CBP when the APACHE II score was 20-29 (P<0.05). Furthermore, CBP treatment significantly decreased the fatality rate and length of stay at the intensive care unit (ICU) for critically ill patients with an APACHE II score of 20-29 (P<0.05). In conclusion, CBP significantly decreases the inflammatory response, shortens the length of stay at the ICU and improves the prognosis for critically ill patients with an APACHE II score of 20-29 points. This observation suggests that the APACHE II score is an important clinical indicator to determine the potential benefit of CBP therapy in critically ill patients.

Role of transforming growth factor ß1 in the inhibition of gastric cancer cell proliferation by melatonin in vitro and in vivo.

Transforming growth factor ß (TGF­ß) is a polypeptide growth factor with various biological activities, and is widely distributed in various tissues. In mammals, TGF­ß has three isoforms: TGF­ß1, 2, and 3, of which TGF­ß1 is most abundant in the TGF­ß family. TGF­ß1 is closely related to the occurrence and development of tumors. A large number of previous studies have shown that melatonin can inhibit a variety of malignancies. Thus, the aim of the present study was to investigate the role of TGF­ß1 in the melatonin­mediated inhibition of the proliferation of gastric cancer cells in vitro and in vivo. TGF­ß1 cytokine stimulation, anti­TGF­ß1 neutralizing antibody blocking, siRNA TGF­ß1 and other means were utilized to explore the role of TGF­ß1 during the course of anti­gastric cancer by melatonin. The results showed that melatonin upregulated the expression of TGF­ß1 in tumor tissues during the process of inhibiting gastric cancer tumor growth in vivo. Melatonin inhibited the proliferation of gastric cancer cells in vitro, accompanied by increased expression of TGF­ß1 in a time­dependent manner. siRNA­mediated silencing of TGF­ß1 and anti­TGF­ß1 neutralizing antibody completely blocked the TGF­ß1 pathway, which significantly antagonized the melatonin­mediated inhibition of the growth and proliferation of gastric cancer cells, and promoted G1 phase to S phase transformation of MFC cells. Our findings suggest that TGF­ß1 is involved in the regulation of the proliferation of tumor cells. One of the ways in which melatonin inhibits the proliferation of gastric cancer cells is dependent on the TGF­ß1 signaling pathway.

Downregulation of AKT and MDM2, Melatonin Induces Apoptosis in AGS and MGC803 Cells.

Melatonin, a neurohormone secreted by the pineal gland, has a variety of biological functions, such as circadian rhythms regulation, anti-oxidative activity, immunomodulatory effects, and anittumor, etc. At present, its antitumor effect has attracted people's attention due to its extensive tissue distribution, good tissue compatibility, and low toxic and side effects. In the gastrointestinal tract, there is high level of melatonin and many studies showed melatonin has effects of anti-gastric cancer. In this experiment, human gastric cancer cell lines AGS and MGC803 were used to investigate the intracellular molecular mechanism of melatonin against gastric cancer. After AGS and MGC803 have been treated with melatonin, the changes of cell morphology and cellular structure were observed under electron microscope. Flow cytometer and apoptosis detection kits were used to analyze the effect of apoptosis on AGS and MGC803. The alterations of apoptosis-related proteins Caspase 9, Caspase 3, and upstream regulators AKT, MDM2 including expression, phosphorylation, and activation were detected to analyze the intracellular molecular mechanism of melatonin inhibiting gastric cancer. In AGS and MGC803 cells with melatonin exposure, cleaved Caspase 9 was upregulated and Caspase 3 was activated; moreover, MDM2 and AKT expression and phosphorylation were downregulated. Melatonin promoted apoptosis of AGS and MGC803 cells by the downregulation of AKT and MDM2. Anat Rec, 302:1544-1551, 2019. © 2019 American Association for Anatomy.

Melatonin induces the apoptosis and inhibits the proliferation of human gastric cancer cells via blockade of the AKT/MDM2 pathway.

Globally, gastric cancer (GC) is one of the most common types of cancer and the third leading cause of cancer­related death. In China, gastric and liver cancers have the highest mortality rates. Melatonin, also known as N-acetyl­5-methoxytryptamine, is a hormone that is produced by the pineal gland in animals and regulates sleep and wakefulness. Melatonin has been shown to inhibit various carcinomas, including GC. There are many different hypotheses to explain the anticancer effects of melatonin, including stimulation of apoptosis, inhibition of cell growth, regulation of anticancer immunity, induction of free-radical scavenging, and the competitive inhibition of estrogen. However, the underlying mechanism by which these effects are elicited remains elusive. The aim of the present study was to investigate the effects of melatonin on human GC cells and determine the underlying molecular mechanism. We treated SGC-7901 GC cells with melatonin and analyzed the resulting protein changes using protein chip technology. Several proteins related to cell apoptosis and proliferation were identified and further tested in SGC-7901 GC cells. We found that melatonin induced cell cycle arrest and the downregulation of CDC25A, phospho-CDC25A (at Ser75), p21 (p21Cip1/p21Waf1) and phospho-p21 (at Thr145). Melatonin also induced upregulation of Bax, downregulation of Bcl-xL, an increase in cleaved caspase-9 level and activation of caspase-3, which confirmed the involvement of the mitochondria in melatonin­induced apoptosis. Upstream regulators of the above proteins, MDM2, phospho-MDM2 (at Ser166) and AKT, phospho-AKT (at Thr308) were all attenuated by melatonin, which led to an increase in p53. The present study demonstrated that the oncostatic effects of melatonin on SGC-7901 GC cells are mediated via the blockade of the AKT/MDM2 intracellular pathway.

Melatonin downregulates nuclear receptor RZR/RORγ expression causing growth-inhibitory and anti-angiogenesis activity in human gastric cancer cells in vitro and in vivo.

An adequate supply of oxygen and nutrients, derived from the formation of novel blood vessels, is critical for the growth and expansion of tumor cells. It has been demonstrated that melatonin (MLT) exhibits marked in vitro and in vivo oncostatic activities. The primary purpose of the present study was to evaluate the in vitro and in vivo antitumor activity of MLT on the growth and angiogenesis of gastric cancer cells, and explore the underlying molecular mechanisms. The present results revealed that MLT inhibited the growth of gastric cancer SGC-7901 cells in a dose- and time-dependent manner. In addition, the present study demonstrated that low concentrations (0.01, 0.1 and 1 mM) of MLT had no clear effect on vascular endothelial growth factor (VEGF) secretion, whereas a high concentration (3 mM) of MLT suppressed VEGF secretion in SGC-7901 cells. Notably, administration of MLT caused suppression of gastric cancer growth and blockade of tumor angiogenesis in tumor-bearing nude mice. Furthermore, MLT treatment reduced the expression of the MLT nuclear receptor RZR/RORγ, SUMO-specific protease 1, hypoxia-inducible factor-1α and VEGF at transcriptional and translational levels within gastric cancer cells during tumorigenesis. In conclusion, MLT nuclear receptor RZR/RORγ may be of great importance in the MLT mediated anti-angiogenesis and growth-inhibitory effect in gastric cancer cells. Since RZR/RORγ is overexpressed in multiple human cancers, MLT may be a promising agent for the treatment of cancers.

Involvement of nuclear receptor RZR/RORγ in melatonin-induced HIF-1α inactivation in SGC-7901 human gastric cancer cells.

The melatonin nuclear receptor is an orphan member of the nuclear receptor superfamily RZR/ROR, which consists of three subtypes (α, ß and γ), suggesting that immunomodulatory and antitumor effects through the intracellular action of melatonin depend on nuclear signaling. In the present study, the biological mechanisms of melatonin were elucidated in association with the RZR/RORγ pathway in SGC-7901 human gastric cancer cells under hypoxia. Melatonin suppressed the activity of RZR/RORγ and SUMO-specific protease 1 (SENP1) signaling pathway, which is essential for stabilization of hypoxia­inducible factor-1α (HIF­1α) during hypoxia. Furthermore, melatonin inhibited the stability of HIF-1α in a time- and conce-ntration-dependent manner in SGC-7901 human gastric cancer cells during hypoxia. Consistently, siRNA-RZR/RORγ effectively blocked the expression of SENP1, HIF-1α and vascular endothelial growth factor (VEGF) production in SGC-7901 cells under hypoxia, suggesting the role of nuclear receptor RZR/RORγ in melatonin-inhibited HIF-1α and VEGF accumulation. Moreover, siRNA RZR/RORγ obviously antagonized to inhibit the action of the gastric cancer cell proliferation by melatonin. Our findings suggest that melatonin suppresses HIF-1α accumulation and VEGF generation via inhibition of melatonin nuclear receptor RZR/RORγ in SGC-7901 cells under hypoxia.

[Anti-gastric cancer effect of melatonin and Bcl-2, Bax, p21 and p53 expression changes].

In order to investigate the role of melatonin in inhibiting the proliferation of murine gastric cancer and the underlying molecular mechanism, we performed an in vivo study by inoculating murine foregastric carcinoma (MFC) cells in mice, and then tumor-bearing mice were treated with different concentrations of melatonin (i.p.). The changes of Bcl-2, Bax, p21 and p53 expressions in tumor tissue were detected by using real-time fluorescence quantitative RT-PCR and Western blot. We found that: (1) melatonin resulted in reductions of tumor's volume and weight in the gastric cancer-bearing mice and thus showed anti-cancer effect; (2) melatonin reduced Bcl-2 expression, but increased the expression of Bax, p53 and p21 in tumor tissue. Our results suggest that melatonin could inhibit the growth of tumors in gastric cancer-bearing mice through accelerating the apoptosis of tumor cells.

Growth-inhibitory activity of melatonin on murine foregastric carcinoma cells in vitro and the underlying molecular mechanism.

Melatonin (MLT) is an indolic hormone produced mainly by the pineal gland. Recent human and animal studies have shown that MLT exerts obvious oncostatic activity both in vitro and in vivo. The purpose of this study was to investigate the antiproliferative effect of MLT on the murine foregastric carcinoma (MFC) cell and to determine the underlying molecular mechanism. Cell viability was determined using the Cell Counting Kit-8 (CCK-8) and the results revealed that MLT exhibited a dose- and time-dependent inhibitory effect on MFC cell growth. Our studies also demonstrated upregulation of p21 and Bax and downregulation of Bcl-2 at both the mRNA and the protein levels in response to MLT treatment of MFC cells. These changes in the expression of these molecules were consistent with the results of the CCK-8. Furthermore, the mRNA and protein expression of membranous MLT receptors was also upregulated. Taken together, these results confirm the oncostatic effect of MLT in MFC cells and the expression of membranous MLT receptors is a potential approach to tumor cells in gastric cancer therapeutic treatment.

Role of CD4+ CD25+ regulatory T cells in melatonin-mediated inhibition of murine gastric cancer cell growth in vivo and in vitro.

Melatonin is an important immune modulator with antitumor functions, and increased CD4(+) CD25(+) regulatory T cells (Tregs) have been observed in tumor tissues of patients and animal models with gastric cancer. However, the relationship between melatonin and Tregs remains unclear. To explore this potential connection, we performed an in vivo study by inoculating the murine foregastric carcinoma (MFC) cell line in mice and then treated them with different doses of melatonin (0, 25, 50, and 100 mg/kg, i.p.) for 1 week. The results showed that melatonin could reduce the tumor tissue and decrease Tregs numbers and Forkhead box p3 (Foxp3) expression in the tumor tissue. An in vitro study was also performed to test the effects of purified Tregs on melatonin-mediated inhibition of MFC cells. The cell cultures were divided into three groups: 1) MFC+ Tregs; 2) MFC only; and 3) MFC+CD4(+) CD25(-) T cells. After treatment with different concentrations of melatonin (0, 2, 4, 6, 8, and 10 mM) for 24 h, a dose-dependent apoptosis and cell cycle arrest at the G2/M phase was detected in melatonin-treated MFC at melatonin concentration higher than 4 mM. There were no significant differences in the rates of apoptosis and cell cycle distributions of MFC among the three groups. In conclusion, the antigastric cancer effect of melatonin is associated with downregulation of CD4(+) CD25(+) Tregs and its Foxp3 expression in the tumor tissue.