Lactate enhances NMNAT1 lactylation to sustain nuclear NAD+ salvage pathway and promote survival of pancreatic adenocarcinoma cells under glucose-deprived conditions.

The aim of this study was to investigate the underlying molecular mechanism behind the promotion of cell survival under conditions of glucose deprivation by l-lactate. To accomplish this, we performed tissue microarray and immunohistochemistry staining to analyze the correlation between the abundance of pan-Lysine lactylation and prognosis. In vivo evaluations of tumor growth were conducted using the KPC and nude mice xenograft tumor model. For mechanistic studies, multi-omics analysis, RNA interference, and site-directed mutagenesis techniques were utilized. Our findings robustly confirmed that l-lactate promotes cell survival under glucose deprivation conditions, primarily by relying on GLS1-mediated glutaminolysis to support mitochondrial respiration. Mechanistically, we discovered that l-lactate enhances the NMNAT1-mediated NAD+ salvage pathway while concurrently inactivating p-38 MAPK signaling and suppressing DDIT3 transcription. Notably, Pan-Kla abundance was significantly upregulated in patients with Pancreatic adenocarcinoma (PAAD) and associated with poor prognosis. We identified the 128th Lysine residue of NMNAT1 as a critical site for lactylation and revealed EP300 as a key lactyltransferase responsible for catalyzing lactylation. Importantly, we elucidated that lactylation of NMNAT1 enhances its nuclear localization and maintains enzymatic activity, thereby supporting the nuclear NAD+ salvage pathway and facilitating cancer growth. Finally, we demonstrated that the NMNAT1-dependent NAD+ salvage pathway promotes cell survival under glucose deprivation conditions and is reliant on the activity of Sirt1. Collectively, our study has unraveled a novel molecular mechanism by which l-lactate promotes cell survival under glucose deprivation conditions, presenting a promising strategy for targeting lactate and NAD+ metabolism in the treatment of PAAD.

LonP1 Links Mitochondria-ER Interaction to Regulate Heart Function.

Interorganelle contacts and communications are increasingly recognized to play a vital role in cellular function and homeostasis. In particular, the mitochondria-endoplasmic reticulum (ER) membrane contact site (MAM) is known to regulate ion and lipid transfer, as well as signaling and organelle dynamics. However, the regulatory mechanisms of MAM formation and their function are still elusive. Here, we identify mitochondrial Lon protease (LonP1), a highly conserved mitochondrial matrix protease, as a new MAM tethering protein. The removal of LonP1 substantially reduces MAM formation and causes mitochondrial fragmentation. Furthermore, deletion of LonP1 in the cardiomyocytes of mouse heart impairs MAM integrity and mitochondrial fusion and activates the unfolded protein response within the ER (UPRER). Consequently, cardiac-specific LonP1 deficiency causes aberrant metabolic reprogramming and pathological heart remodeling. These findings demonstrate that LonP1 is a novel MAM-localized protein orchestrating MAM integrity, mitochondrial dynamics, and UPRER, offering exciting new insights into the potential therapeutic strategy for heart failure.

Identification of three metabolic subtypes in gastric cancer and the construction of a metabolic pathway-based risk model that predicts the overall survival of GC patients.

Gastric cancer (GC) is highly heterogeneous and GC patients have low overall survival rates. It is also challenging to predict the prognosis of GC patients. This is partly because little is known about the prognosis-related metabolic pathways in this disease. Hence, our objective was to identify GC subtypes and genes related to prognosis, based on changes in the activity of core metabolic pathways in GC tumor samples. Differences in the activity of metabolic pathways in GC patients were analyzed using Gene Set Variation Analysis (GSVA), leading to the identification of three clinical subtypes by non-negative matrix factorization (NMF). Based on our analysis, subtype 1 showed the best prognosis while subtype 3 exhibited the worst prognosis. Interestingly, we observed marked differences in gene expression between the three subtypes, through which we identified a new evolutionary driver gene, CNBD1. Furthermore, we used 11 metabolism-associated genes identified by LASSO and random forest algorithms to construct a prognostic model and verified our results using qRT-PCR (five matched clinical tissues of GC patients). This model was found to be both effective and robust in the GSE84437 and GSE26253 cohorts, and the results from multivariate Cox regression analyses confirmed that the 11-gene signature was an independent prognostic predictor (p < 0.0001, HR = 2.8, 95% CI 2.1-3.7). The signature was found to be relevant to the infiltration of tumor-associated immune cells. In conclusion, our work identified significant GC prognosis-related metabolic pathways in different GC subtypes and provided new insights into GC-subtype prognostic assessment.

WZ35 inhibits gastric cancer cell metastasis by depleting glutathione to promote cellular metabolic remodeling.

This study aimed at elucidating the crosstalk between redox reaction and metabolic remodeling through uncovering the mechanism underlying WZ35-mediated reactive oxygen species (ROS) production and regulation of amino acid metabolism to inhibit gastric cancer (GC) cell metastasis. The activity and biosafety of curcumin analog, WZ35, were verified in vitro and in vivo. The potential molecular mechanism underlying WZ35-mediated enhanced radiotherapeutic sensitivity by reduced Glutathione (GSH) depletion was elucidated by RNA sequencing, single-cell sequencing (scRNA-seq), metabolic mass spectrometry, and other molecular experiments. Compared to curcumin, WZ35 proved more potent anti-proliferative and anti-metastasis properties. Importantly, we demonstrated that WZ35 could consume GSH in multiple ways, including by reduction of raw materials and consumption reserves, inhibition of reformation, and enhanced decomposition. Mechanistically, we identify that WZ35 maintains the GSH depletion phenotype through the ROS-YAP-AXL-ALKBH5-GLS2 loop, further backing the relevance of metabolic remodeling in the tumor microenvironment with tumor metastasis and the role of m6A in tumor metastasis. Collectively, our study identified WZ35 as a novel GSH depletion agent and a previously undiscovered GSH depletion loop mechanism in GC cell metastasis.

SRSF3 and HNRNPH1 Regulate Radiation-Induced Alternative Splicing of Protein Arginine Methyltransferase 5 in Hepatocellular Carcinoma.

Protein arginine methyltransferase 5 (PRMT5) is an epigenetic regulator which has been proven to be a potential target for cancer therapy. We observed that PRMT5 underwent alternative splicing (AS) and generated a spliced isoform PRMT5-ISO5 in hepatocellular carcinoma (HCC) patients after radiotherapy. However, the regulatory mechanism and the clinical implications of IR-induced PRMT5 AS are unclear. This work revealed that serine and arginine rich splicing factor 3 (SRSF3) silencing increased PRMT5-ISO5 level, whereas heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1) silencing reduced it. Then, we found that SRSF3 and HNRNPH1 competitively combined with PRMT5 pre-mRNA located at the region around the 3'- splicing site on intron 2 and the alternative 3'- splicing site on exon 4. IR-induced SRSF3 downregulation led to an elevated level of PRMT5-ISO5, and exogenous expression of PRMT5-ISO5 enhanced cell radiosensitivity. Finally, we confirmed in vivo that IR induced the increased level of PRMT5-ISO5 which in turn enhanced tumor killing and regression, and liver-specific Prmt5 depletion reduced hepatic steatosis and delayed tumor progression of spontaneous HCC. In conclusion, our data uncover the competitive antagonistic interaction of SRSF3 and HNRNPH1 in regulating PRMT5 splicing induced by IR, providing potentially effective radiotherapy by modulating PRMT5 splicing against HCC.

A novel small molecule glycolysis inhibitor WZ35 exerts anti-cancer effect via metabolic reprogramming.

BACKGROUND: Liver cancer is the fifth leading cause of cancer death worldwide, but early diagnosis and treatment of liver cancer remains a clinical challenge. How to screen and diagnose liver cancer early and prolong the survival rate is still the focus of researchers. METHODS: Cell experiments were used to detect the effect of WZ35 on the colony formation ability and proliferation activity of hepatoma cells, nude mouse experiment to observe the in vivo anticancer activity and toxic side effects of WZ35; metabolomics analysis, glucose metabolism experiment and Seahorse analysis of liver cancer cells treated with WZ35; cell experiments combined with bioinformatics analysis to explore the mechanism of WZ35-mediated metabolic reprogramming to exert anticancer activity; tissue microarray and case analysis to evaluate the clinical significance of biomarkers for early diagnosis, treatment and prognosis evaluation of liver cancer. RESULTS: WZ35 inhibited the proliferation activity of various cell lines of liver cancer, and showed good therapeutic effect in nude mice model of hepatocellular carcinoma without obvious toxic and side effects; WZ35 inhibited the absorption of glucose in hepatoma cells, and the drug effect glycolysis, phosphorylation and purine metabolism are relatively seriously damaged; WZ35 mainly inhibits YAP from entering the nucleus as a transcription factor activator by activating oxidative stress in liver cancer cells, reducing the transcription of GLUT1, and finally reducing its GLUT1. Tissue microarray and case analysis showed that GLUT1 and YAP were highly expressed and correlated in liver cancer patients, and were associated with poor patient prognosis. The GLUT1-YAP risk model had a high score in predicting prognosis. CONCLUSION: The study confirms that WZ35 is a small molecule glycolysis inhibitor, and through its properties, it mediates metabolic reprogramming dominated by impaired glycolysis, oxidative phosphorylation and purine metabolism to inhibit the proliferation activity of liver cancer cells. Our findings present novel insights into the pathology of liver cancer and potential targets for new therapeutic strategies. GLUT1-YAP has important reference significance for predicting the stages of disease progression in liver cancer patients and have the potential to serve as novel biomarkers for the diagnosis and treatment of liver cancer.

Cold protection allows local cryotherapy in a clinical-relevant model of traumatic optic neuropathy.

Therapeutic hypothermia (TH) is potentially an important therapy for central nervous system (CNS) trauma. However, its clinical application remains controversial, hampered by two major factors: (1) Many of the CNS injury sites, such as the optic nerve (ON), are deeply buried, preventing access for local TH. The alternative is to apply TH systemically, which significantly limits the applicable temperature range. (2) Even with possible access for 'local refrigeration', cold-induced cellular damage offsets the benefit of TH. Here we present a clinically translatable model of traumatic optic neuropathy (TON) by applying clinical trans-nasal endoscopic surgery to goats and non-human primates. This model faithfully recapitulates clinical features of TON such as the injury site (pre-chiasmatic ON), the spatiotemporal pattern of neural degeneration, and the accessibility of local treatments with large operating space. We also developed a computer program to simplify the endoscopic procedure and expand this model to other large animal species. Moreover, applying a cold-protective treatment, inspired by our previous hibernation research, enables us to deliver deep hypothermia (4 °C) locally to mitigate inflammation and metabolic stress (indicated by the transcriptomic changes after injury) without cold-induced cellular damage, and confers prominent neuroprotection both structurally and functionally. Intriguingly, neither treatment alone was effective, demonstrating that in situ deep hypothermia combined with cold protection constitutes a breakthrough for TH as a therapy for TON and other CNS traumas.

Hypothermic therapy is a radical type of treatment that involves cooling a person's core body temperature several degrees below normal to protect against brain damage. Lowering body temperature slows blood flow, which reduces inflammation, and eases metabolic demands, similar to hibernation. It can also reduce lasting damage to the brain and aid recovery when used to treat people who have gone into cardiac arrest, where their heart suddenly stops beating. Recently, there has been renewed interest in using hypothermic therapy to treat people who have sustained traumatic brain injuries, which can cause brain swelling, and other nerve injuries. However, its use remains controversial because clinical trials have failed to show that inducing mild hypothermia provides any benefit for people with severe nerve injuries. This might be because cooling cells to near-freezing temperatures can damage their internal structural supports, called microtubules, thwarting any therapeutic benefit. Traumatic optical neuropathy is a type of injury in which the optic nerve ­ the nerve that connects the eyes to the brain ­ is damaged or severed, causing vision loss. There is currently no clinically proven treatment for this condition, nor is there a system that can test local treatments in large animals as a prior test to using the treatment in the clinic. Therefore, Zhang et al. wanted to establish such a animal model and test whether local hypothermic therapy could help protect the optic nerve. Zhang et al. used a surgical tool guided by an endoscope (a thin plastic tube with a light and camera attached to it) to injure the optic nerves of goats, and then deliver hypothermic therapy. To cool the surgically-injured nerves to a chilly 4C, Zhang et al. applied a deep-cooling agent, using a second reagent (a cocktail of protease inhibitors) to protect the cells' microtubules from cold-induced damage, an insight gained from a previous study of hibernating animals. This was critical, as the hypothermic therapy was only effective when the secondary protective agent was applied. The combination therapy developed by Zhang et al. relieved some aspects of nerve degeneration at the injury site and activated an anti-inflammatory response in cells, but did not restore vision. To simplify surgical techniques, Zhang et al. also developed a computer program which generates virtual surgical paths for up-the-nose endoscopic procedures based on brain scans of an animal's skull. This program was successfully applied in a range of large animals, including goats and macaque monkeys. Zhang et al.'s work establishes a method to study treatments for traumatic optical neuropathy using large animals, including hypothermic therapy. The methods developed could also be useful to study other optic nerve disorders, such as optic neuritis or ischemic optic neuropathy.

Curcumin micelles suppress gastric tumor cell growth by upregulating ROS generation, disrupting redox equilibrium and affecting mitochondrial bioenergetics.

Gastric cancer is the fourth most common cancer and the second most frequent cause of cancer death worldwide. Chemotherapy is an important treatment. However, traditional chemotherapy drugs have low bioavailability and targeting ability. Therefore, we developed curcumin-encapsulated micelles for the treatment of gastric cancer and investigated their antitumor efficacy and active mechanism. Gastric cancer cells were treated with different concentrations of curcumin micelles. MTS cell proliferation assays, flow cytometry (FCM), real time cellular analysis (RTCA) and nude mice xenografts were used to evaluate the effects of curcumin micelles on gastric cancer cell growth in vitro and in vivo. Western blotting was performed to analyze the protein levels of the indicated molecules. A Seahorse bioenergetics analyzer was used to investigate alterations in oxygen consumption and the aerobic glycolysis rate. Curcumin micelles significantly inhibited proliferation and colony formation and induced apoptosis in gastric tumor cells compared to the control groups. We further investigated the mechanism of curcumin micelles on gastric tumor cells and demonstrated that curcumin micelles acted on mitochondrial proteins, causing changes in mitochondrial function and affecting mitochondrial bioenergetics. Furthermore, curcumin micelles decreased mitochondrial membrane potential, increased reactive oxygen species (ROS) generation and disrupted redox equilibrium. The nude mouse model verified that curcumin micelle treatment significantly attenuated tumor growth in vivo. Curcumin micelles suppress gastric tumor cell growth in vitro and in vivo. The mechanism may be related to increasing ROS generation, disrupting redox equilibrium and affecting mitochondrial bioenergetics.

The curcumin analogue WZ35 affects glycolysis inhibition of gastric cancer cells through ROS-YAP-JNK pathway.

To investigate the anti-tumor activities of WZ35 and its possible molecular mechanism, bioinformatics analysis and the hematoxylin-eosin (HE) staining were applied to evaluate the Yes-associated-protein (YAP) level in gastric cancer. Cell counting kit-8 (CCK-8) was used to examine cell viability. Apoptosis was determined by flow cytometry analysis. Seahorse bioenergetics analyzer was used to investigate the alteration of oxygen consumption and aerobic glycolysis rate. SiRNA transfection was applied to silence endogenous YAP. Western blot was performed to detect indicated proteins. We found that treatment of gastric cancer cells with WZ35 exerted stronger anti-tumor activities than curcumin. Mechanistically, our research showed that WZ35 inhibited glycolysis, and induced reactive oxygen species (ROS) generation, resulting in Jun N-terminal Kinase (JNK) activation through downregulation of YAP in gastric cancer cells. ROS mediated YAP downregulation and JNK activation was regulated by glycolysis. Abrogation of ROS production markedly attenuated WZ35 induced anti-tumor activities as well as YAP downregulation and JNK activation. Similarly, the JNK inhibitor significantly reversed WZ35 induced anti-tumor activities in gastric cancer cells. Our study reveals a novel anti-gastric cancer mechanism of WZ35 by inhibiting glycolysis through the ROS-YAP-JNK pathway. WZ35 might be a potential therapeutics for the treatment of gastric cancer.

Curcumin derivative WZ35 inhibits tumor cell growth via ROS-YAP-JNK signaling pathway in breast cancer.

BACKGROUND: Breast cancer is the most prevalent cancer among women worldwide. WZ35, an analog of curcumin, has been demonstrated to remarkably improve the pharmacokinetic profiles in vivo compared with curcumin. WZ35 exhibits promising antitumor activity in gastric cancer, HCC, colon cancer. However, antitumor effects of WZ35 in breast cancer and its underlying molecular mechanisms remain unclear. METHODS: CCK8, Flow cytometry and transwell assays were used to measure cell proliferation, cell cycle arrest, apoptosis, cell migration and invasion. We constructed xenograft mouse model and lung metastasis model to assess the antitumor activities of WZ35 in vivo. To explore the underlying molecular mechanisms of WZ35, we performed a series of overexpression and knockdown experiments. The cellular oxygen consumption rates (OCRs) was measured to assess mitochondrial dysfunction. RESULTS: We found that treatment of breast cancer cells with WZ35 exerts stronger anti-tumor activities than curcumin both in vitro and in vivo. Mechanistically, our research showed that WZ35 induced reactive oxygen species (ROS) generation and subsequent YAP mediated JNK activation in breast cancer cells. Abrogation of ROS production markedly attenuated WZ35 induced anti-tumor activities as well as YAP and JNK activation. In addition, ROS mediated YAP and JNK activation induced mitochondrial dysfunction in breast cancer cells. CONCLUSION: Our study showed that novel anti-cancer mechanisms of WZ35 in breast cancer cells and ROS-YAP-JNK pathway might be a potential therapeutic target for the treatment of breast cancer patients.

Mechanisms Underlying Therapeutic Effects Of Traditional Chinese Medicine On Gastric Cancer.

Gastric cancer (GC) is one of the most common malignant tumors in the world. It is the fourth most common cancer and has the second highest mortality rate globally. Metastasis is an important feature of gastric cancer and is the most common cause of death. Exploring the mechanism underlying the metastasis of gastric cancer and searching for new drug targets has become the focus of several studies. Traditional Chinese medicine may show promise for treatment of gastric cancer. In this review, we report the recent progress in research on the anti-metastasis activity of Chinese medicine, to facilitate clinical development of treatments for gastric cancer.

A curcumin derivative, WZ35, suppresses hepatocellular cancer cell growth via downregulating YAP-mediated autophagy.

Hepatocellular carcinoma (HCC) is a common cancer type throughout the world. Due to the high occurrence rate and mortality, liver cancer is one of the leading causes of cancer associated death. With the development of monoclonal antibodies and immunotherapy, the mortality of HCC cancer patients has reduced. However, the recurrence and outcomes of patients remain poor. Therefore, there is an urgent need to develop more effective drugs for HCC therapy. WZ35, a novel curcumin derivative, exhibits potential anti-tumor activity in gastric cancer cells by regulating ROS dependent JNK activation and ER stress. Here, we evaluated the tumor suppressive activity of WZ35 in hepatocellular carcinoma in vitro and in vivo. CCK-8 was used to detect cell viability with or without curcumin or WZ35; cell apoptosis was determined by flow cytometry analysis; GFP-LC3 plasmids were used to investigate the level of autophagy-associated LC3; siRNA transfection was applied to silence endogenous YAP; and western blot was performed to detect the alteration of indicated molecules. Bioinformatics analysis and IHC assay were applied to evaluate the YAP level in normal and liver cancer tissues. In this study, we found that WZ35 effectively suppresses HCC cancer cell growth in vitro and in vivo by promoting cell apoptosis. Importantly, downregulation of YAP contributes to WZ35 caused autophagy inhibition which is different from that of curcumin. We also confirmed that WZ35 is more effective at suppressing HCC cell growth in vivo. Finally, we confirmed that YAP was significantly overexpressed in liver cancer tissues. Collectively, these data indicate that WZ35 could be considered as a promising compound for HCC therapy.

Rapamycin inhibits proliferation and induces autophagy in human neuroblastoma cells.

Objective To investigate the effect of Rapamycin on proliferation and autophagy in human neuroblastoma (NB) cell lines and to elucidate the possible mechanism. Methods NB cells were treated with different concentrations of Rapamycin. Cell counting kit-8 (CCK-8) was used to measure proliferation, and flow cytometry (FCM) was used to analyze the cell cycle. EM was used to observe cell morphological changes. Western blotting (WB) was performed to detect the expression of Beclin-1, LC3-I/II, P62, mammalian target of Rapamycin (mTOR), and p-mTOR. Results Rapamycin inhibited the spread of NB cells in a dose- and time-dependent manner and arrested the cell cycle at the G0/G1 phase. EM showed autophagosomes in NB cells treated with Rapamycin. The WB results showed that the expression levels of Beclin-1 and LC3-II/LC3-I were significantly elevated in NB cells treated with Rapamycin, while the expression levels of P62, mTOR, and p-mTOR proteins were significantly reduced compared with the control cells (P<0.05). Conclusion Rapamycin inhibits cell proliferation and induces autophagy in human NB cell lines. The mechanism may be related to suppression of the mTOR signaling pathway.

The curcumin derivative WZ35 activates ROS-dependent JNK to suppress hepatocellular carcinoma metastasis.

Tumor metastasis is the leading cause of cancer death; due to the progress made in the elucidation of the mechanism of cancer cell metastasis, there is hope for patients with severe stages of cancer. Curcumin, as a novel anti-cancer drug, has been applied in cancer therapy; however, the toxicity of curcumin hinders its application. Herein, we constructed a novel derivative, WZ35, and evaluated its metastatic inhibition properties in vitro and in vivo. CCK-8 assay was performed to evaluate the tumor suppressive activity of WZ35. Cell apoptosis was detected by flow cytometry analysis. Transwell cell migration assay and RTCA were used to detect cell migration in mock and WZ35-treated cells. Western blotting was performed to analyze molecular alteration with different treatments. In this study, we found that curcumin and its derivative WZ35 could dramatically suppress proliferation, invasion, and migration of the hepatocellular HCCLM3, HepG2, and Huh7 cancer cells. Moreover, the cancer cell metastatic markers MMP-2, MMP-9, and N-cadherin were decreased, and E-cadherin was up-regulated. In addition, our data show that WZ35 promotes ROS-dependent JNK activation that is essential for WZ35-caused cell metastasis suppression. Moreover, the NAC and JNK inhibitor SP600125 could dramatically reverse WZ35-caused MMP-2, MMP-9, and N-cadherin reduction and E-cadherin up-regulation. We have also found that WZ35 exhibits powerful anti-metastasis activity of HCCLM3 in vivo. In conclusion, our data indicated that WZ35 could be a candidate for the treatment of metastatic liver cancer patients.

Dicer regulates non-homologous end joining and is associated with chemosensitivity in colon cancer patients.

DNA double-strand break (DSB) repair is an important mechanism underlying chemotherapy resistance in human cancers. Dicer participates in DSB repair by facilitating homologous recombination. However, whether Dicer is involved in non-homologous end joining (NHEJ) remains unknown. Here, we addressed whether Dicer regulates NHEJ and chemosensitivity in colon cancer cells. Using our recently developed NHEJ assay, we found that DSB introduction by I-SceI cleavage leads to Dicer upregulation. Dicer knockdown increased SIRT7 binding and decreased the level of H3K18Ac (acetylated lysine 18 of histone H3) at DSB sites, thereby repressing the recruitment of NHEJ factors to DSB sites and inhibiting NHEJ. Dicer overexpression reduced SIRT7 binding and increased the level of H3K18Ac at DSB sites, promoting the recruitment of NHEJ factors to DSBs and moderately enhancing NHEJ. Dicer knockdown and overexpression increased and decreased, respectively, the chemosensitivity of colon cancer cells. Dicer protein expression in colon cancer tissues of patients was directly correlated with chemoresistance. Our findings revealed a function of Dicer in NHEJ-mediated DSB repair and the association of Dicer expression with chemoresistance in colon cancer patients.

Curcumin suppresses gastric tumor cell growth via ROS-mediated DNA polymerase γ depletion disrupting cellular bioenergetics.

BACKGROUND: Curcumin, as a pro-apoptotic agent, is extensively studied to inhibit tumor cell growth of various tumor types. Previous work has demonstrated that curcumin inhibits cancer cell growth by targeting multiple signaling transduction and cellular processes. However, the role of curcumin in regulating cellular bioenergetic processes remains largely unknown. METHODS: Western blotting and qRT-PCR were performed to analyze the protein and mRNA level of indicated molecules, respectively. RTCA, CCK-8 assay, nude mice xenograft assay, and in vivo bioluminescence imaging were used to visualize the effects of curcmin on gastric cancer cell growth in vitro and in vivo. Seahorse bioenergetics analyzer was used to investigate the alteration of oxygen consumption and aerobic glycolysis rate. RESULTS: Curcumin significantly inhibited gastric tumor cell growth, proliferation and colony formation. We further investigated the role of curcumin in regulating cellular redox homeostasis and demonstrated that curcumin initiated severe cellular apoptosis via disrupting mitochondrial homeostasis, thereby enhancing cellular oxidative stress in gastric cancer cells. Furthermore, curcumin dramatically decreased mtDNA content and DNA polymerase γ (POLG) which contributed to reduced mitochondrial oxygen consumption and aerobic glycolysis. We found that curcumin induced POLG depletion via ROS generation, and POLG knockdown also reduced oxidative phosphorylation (OXPHOS) activity and cellular glycolytic rate which was partially rescued by ROS scavenger NAC, indiating POLG plays an important role in the treatment of gastric cancer. Data in the nude mice model verified that curcumin treatment significantly attenuated tumor growth in vivo. Finally, POLG was up-regulated in human gastric cancer tissues and primary gastric cancer cell growth was notably suppressed due to POLG deficiency. CONCLUSIONS: Together, our data suggest a novel mechanism by which curcumin inhibited gastric tumor growth through excessive ROS generation, resulting in depletion of POLG and mtDNA, and the subsequent disruption of cellular bioenergetics.

EF24 induces ROS-mediated apoptosis via targeting thioredoxin reductase 1 in gastric cancer cells.

Gastric cancer (GC) is one of the leading causes of cancer mortality in the world, and finding novel agents for the treatment of advanced gastric cancer is of urgent need. Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. Although EF24 demonstrates potent anticancer effïcacy in numerous types of human cancer cells, the cellular targets of EF24 have not been fully defined. We report here that EF24 may interact with the thioredoxin reductase 1 (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme, to induce reactive oxygen species (ROS)-mediated apoptosis in human gastric cancer cells. By inhibiting TrxR1 activity and increasing intracellular ROS levels, EF24 induces a lethal endoplasmic reticulum stress in human gastric cancer cells. Importantly, knockdown of TrxR1 sensitizes cells to EF24 treatment. In vivo, EF24 treatment markedly reduces the TrxR1 activity and tumor cell burden, and displays synergistic lethality with 5-FU against gastric cancer cells. Targeting TrxR1 with EF24 thus discloses a previously unrecognized mechanism underlying the biological activity of EF24, and reveals that TrxR1 is a good target for gastric cancer therapy.

Selective killing of gastric cancer cells by a small molecule targeting ROS-mediated ER stress activation.

Gastric cancer is one of the leading causes of cancer mortality in the world. Curcumin is a natural product with multiple pharmacological activities, while its clinical application has been limited by the poor chemical stability. We have previously designed a series of curcumin derivatives with high stability and anticancer potentials. The present study aims to identify the anti-cancer effects and mechanisms of WZ26, an analog of curcumin, in gastric cancer cells. In vitro, WZ26 showed higher chemical stability and much stronger anti-proliferative effects than curcumin, accompanied by dose-dependent induction of cell cycle arrest and apoptosis in gastric cancer cells. Mechanistically, the novel compound WZ26 induced ROS production, resulting in the activation of JNK-mitochondrial and ER stress apoptotic pathways. Blockage of ROS production totally reversed WZ26-induced JNK activation, Bcl-2/Bax decrease, ER stress activation, and final cell apoptosis in SGC-7901 cells. WZ26 also exhibited potent anti-tumor effects in human gastric cancer cell xenograft models. WZ26 could be considered as a potential chemotherapeutic agent for the treatment of advanced gastric cancer. In addition, this study also demonstrated that ROS production could be act as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial and ER stress-related death pathway. © 2015 Wiley Periodicals, Inc.

[Protective effect of tadalafil against ischemia-reperfusion injury in rats].

OBJECTIVE: To investigate the protective effect of phosphodiesterase type 5 inhibitors (tadalafil) on the testis following testicular ischemia-reperfusion injury in rats. METHODS: Eighty-four healthy adult male SD rats were randomly and equally divided into groups A (sham operation), B (testicular torsion + low-dose tadalafil), C (testicular torsion + high-dose tadalafil), and D (testicular torsion + placebo). Models were established in the latter three groups by 7200 torsion of the right testis for 2 hours. The animals in groups A and B were treated by gavage with tadalafil at the dose of 0. 5 mg per kg per day, those in group C at 2 mg per kg per day, and those in group D with saline at the same dose. After 3, 7, and 14 days of treatment, the torsioned testes were harvested for evaluation of the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the testis tissue. The pathological changes in the testis were observed under the light microscope. RESULTS: At 3, 7, and 14 days, the SOD activity was (254.46 +/- 7.43), (278.49 +/- 8.33), and (317.99 +/- 3.31) nU/mg prot in group B, and (277.12 +/- 8.80), (309.40 +/- 2.14), and (320.39 +/- 4.72) nU/mg prot in group C, all obviously higher than in D ([223.21 +/- 4.65], [231.45 +/- 4.16] and [248.28 +/- 5.74] nU/mg prot), while the MDA content was lower in the former two groups than in the latter. At 3 and 7 days, the SOD activity was significantly higher and the MDA level significantly lower in group C than in B (both P < 0.01) , while at 14 days, neither showed any remarkable differences between the two groups (P > 0.05). No obvious histopathological change was observed in the testis tissue of group A. At 3 and 7 days, pathological examination of the testis tissue revealed significant differences in the number of seminiferous epithelial layers, testicular histological score, and seminiferous tubule diameter in group B (P < 0.01), but the three indexes at 14 days in group B and at 7 days in group C exhibited no remarkable differences from those at 14 days in group A. CONCLUSION: Tadalafil can alleviate testicular ischemia-reperfusion injury following testis torsion/detorsion in a time- and dose-dependent manner.

Preparation of curcumin micelles and the in vitro and in vivo evaluation for cancer therapy.

Poor water solubility as well as poor bioavailability of curcumin has greatly hindered its applications in cancer therapy. In the present study, a highly water soluble curcumin nano-formulation was developed and its in vivo anti-cancer efficiency in nude BALB/c mouse model was evaluated. Unlike native curcumin, the developed curcumin micelles were quickly dissolved into aqueous solution with significant improvement of water solubility (-10(4) fold increasement compared to its native form). The developed curcumin micelles had a narrow size distribution (18-28 nm) with high drug encapsulation efficiency (85%-95%). The developed curcumin micelles were characterized by X-raydiffraction (XRD), Differential scanning calorimetric (DSC), and Fluorescence spectral analysis. We observed the enhanced stability of curcumin in micelles formulation in phosphate buffer solution (PBS). In vitro cytotoxicity assay indicated that the curcumin micelles was comparatively more effective than native curcumin against various cancer cell lines due to the enhanced cellular uptake of curcumin yet resulting in the apoptosis of cancer cells. Western blotting study revealed that the induction apoptosis of S-65 cancer cells by curcumin micelles was mainly due to the down-regulation of p-Rb, Blc-2, p-AKT expression and caspase-9 activation. In vivo anti-tumor test in nude BALB/c mouse bearing S-65 xenografts indicated the intraperitoneal injection of curcumin micelles (25 mg/kg) could significantly inhibit tumor growth as compared with native curcumin treatment (p < 0.05), which was accompanied by significantly increased apoptosis of tumor cells and diminished vascular endothelial growth factor expression in tumor tissue (p < 0.05).