Tanshinone IIA reverses gefitinib resistance in EGFR-mutant lung cancer via inhibition of SREBP1-mediated lipogenesis.

BACKGROUND AND AIM: Gefitinib resistance is an urgent problem to be solved in the treatment of non-small cell lung cancer (NSCLC). Tanshinone IIA (Tan IIA) is one of the main active components of Salvia miltiorrhiza, which exhibits significant antitumor effects. The aim of this study is to explore the reversal effect of Tan IIA on gefitinib resistance in the epidermal growth factor receptor (EGFR)-mutant NSCLC and the underlying mechanism. EXPERIMENTAL PROCEDURE: CCK-8, colony formation assay, and flow cytometry were applied to detect the cytotoxicity, proliferation, and apoptosis, respectively. The changes in lipid profiles were measured by electrospray ionization-mass spectrometry (MS)/MS. Western blot, real-time q-PCR, and immunohistochemical were used to detect the protein and the corresponding mRNA levels. The in vivo antitumor effect was validated by the xenograft mouse model. KEY RESULTS: Co-treatment of Tan IIA enhanced the sensitivity of resistant NSCLC cells to gefitinib. Mechanistically, Tan IIA could downregulate the expression of sterol regulatory element binding protein 1 (SREBP1) and its downstream target genes, causing changes in lipid profiles, thereby reversing the gefitinib-resistance in EGFR-mutant NSCLC cells in vitro and in vivo. CONCLUSIONS AND IMPLICATIONS: Tan IIA improved gefitinib sensitivity via SREBP1-mediated lipogenesis. Tan IIA could be a potential candidate to enhance sensitivity for gefitinib-resistant NSCLC patients.

Paraptosis: a non-classical paradigm of cell death for cancer therapy.

Due to the sustained proliferative potential of cancer cells, inducing cell death is a potential strategy for cancer therapy. Paraptosis is a mode of cell death characterized by endoplasmic reticulum (ER) and/or mitochondrial swelling and cytoplasmic vacuolization, which is less investigated. Considerable evidence shows that paraptosis can be triggered by various chemical compounds, particularly in cancer cells, thus highlighting the potential application of this non-classical mode of cell death in cancer therapy. Despite these findings, there remain significant gaps in our understanding of the role of paraptosis in cancer. In this review, we summarize the current knowledge on chemical compound-induced paraptosis. The ER and mitochondria are the two major responding organelles in chemical compound-induced paraptosis, which can be triggered by the reduction of protein degradation, disruption of sulfhydryl homeostasis, overload of mitochondrial Ca2+, and increased generation of reactive oxygen species. We also discuss the stumbling blocks to the development of this field and the direction for further research. The rational use of paraptosis might help us develop a new paradigm for cancer therapy.

Lipidomics reveals that sustained SREBP-1-dependent lipogenesis is a key mediator of gefitinib-acquired resistance in EGFR-mutant lung cancer.

Patients with EGFR mutations in non-small cell lung cancer (NSCLC) have been greatly benefited from gefitinib, however, the therapeutic has failed due to the presence of acquired resistance. In this study, we show that gefitinib significantly induces downregulation of Sterol Regulator Element Binding (SREBP1) in therapy-sensitive cells. However, this was not observed in EGFR mutant NSCLC cells with acquired resistance. Lipidomics analysis showed that gefitinib could differently change the proportion of saturated phospholipids and unsaturated phospholipids in gefitinib-sensitive and acquired-resistant cells. Besides, levels of ROS and MDA were increased upon SREBP1 inhibition and even more upon gefitinib treatment. Importantly, inhibition of SREBP1 sensitizes EGFR-mutant therapy-resistant NSCLC to gefitinib both in vitro and in vivo models. These data suggest that sustained de novo lipogenesis through the maintenance of active SRBEP-1 is a key feature of acquired resistance to gefitinib in EGFR mutant lung cancer. Taken together, targeting SREBP1-induced lipogenesis is a promising approach to overcome acquired resistance to gefitinib in EGFR-mutant lung cancer.

Cryptotanshinone strengthens the effect of gefitinib against non-small cell lung cancer through inhibiting transketolase.

Lung cancer is the leading cause of cancer-related mortality and causes more than a million deaths per year. Gefitinib is the first-line agent of advanced lung cancer, however, resistance to gefitinib becomes a major problem in clinical application. Transketolase (TKT) is a key enzyme functioning between the oxidative arm and the non-oxidative arm of the pentose phosphate pathway. In this study, we firstly found that the expression of TKT was remarkably up-regulated in NSCLC cells, while the knockdown of TKT could inhibit cell proliferation and enhance the effect of gefitinib on NSCLC cells, which indicated the role of TKT in treating advanced lung cancer. Cryptotanshinone (CTS) is a natural active compound possessing anti-cancer effect. Here we demonstrated that CTS could strengthen the effect of gefitinib on NSCLC cells via inhibition of TKT in vitro and in vivo. Moreover, Nrf2 was involved in the repression of CTS on TKT expression. Collectively, these findings indicated the role of TKT in lung cancer progression and may provide novel therapeutic strategies to overcome resistance to gefitinib. Furthermore, CTS may serve as a new candidate in adjuvant treatment of advanced lung cancer.

Comparative pharmacokinetics and tissue distribution of cryptotanshinone, tanshinone IIA, dihydrotanshinone I, and tanshinone I after oral administration of pure tanshinones and liposoluble extract of Salvia miltiorrhiza to rats.

Salvia miltiorrhiza is one of the most commonly used traditional Chinese medicines in the treatment of cardiovascular and cerebrovascular diseases. Cryptotanshinone (CTS), tanshinone IIA (Tan IIA), dihydrotanshinone I (diTan I), and tanshinone I (Tan I) are the main active compounds in the liposoluble extract of Salvia miltiorrhiza. The differences in the pharmacokinetic and tissue distribution behaviors of the four tanshinones after oral administration of the liposoluble extract of Salvia miltiorrhiza and pure compounds are not clear. This study aims to compare the pharmacokinetics and tissue distribution of the four tanshinones after oral administration of pure tanshinone monomers and the liposoluble extract of Salvia miltiorrhiza. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis method was developed for the determination of the four tanshinones. The results showed that the AUC and Cmax of tanshinones in rats receiving the extract of Salvia miltiorrhiza were significantly increased compared with those receiving the pure tanshinones. In the tissue distribution experiments, the AUC of the four tanshinones in the extract was much greater than the AUC of the monomers in the lung, heart, kidney, liver, and brain, and the coexisting constituents particularly promoted the distribution of tanshinones into tissues that the drug cannot sufficiently penetrate. These findings suggested that the coexisting constituents in the liposoluble extract of Salvia miltiorrhiza play an important role in the alteration of plasma concentration and tissue distribution of the four tanshinones. Understanding these differences could be of significance for the development and application of Salvia miltiorrhiza extract and tanshinone components.

Sesamin Enhances Nrf2-Mediated Protective Defense against Oxidative Stress and Inflammation in Colitis via AKT and ERK Activation.

Ulcerative colitis (UC) is a major form of inflammatory bowel disease (IBD) with high incidence and prevalence in many countries. Patients with UC usually suffer from a lifetime of debilitating physical symptoms. Therefore, developing effective therapeutic strategy that can manage this disease better and improve patients' life quality is in urgent need. Sesamin (SSM) is a lignan derived from sesame seeds. In this study, the protective effect of SSM against UC and the underlying mechanism were investigated in vitro and in vivo. Our data showed that SSM protected Caco-2 cells from H2O2-induced oxidative stress injury via GSH-mediated scavenging of reactive oxygen species (ROS). Dual luciferase reporter assay showed that the transcriptional activity of nuclear factor erythroid-related factor 2 (Nrf2) was significantly increased by SSM, and the ability of SSM to activate Nrf2-targeted genes was further confirmed in Caco-2 cells using western blot and quantitative real-time PCR (qRT-PCR). In contrast, Nrf2 knockdown abolished the protective effect of SSM. Additionally, we found that SSM also activated advanced protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in Caco-2 cells, while either AKT or ERK inhibition can prevent SSM-mediated nuclear translocation of Nrf2. Furthermore, SSM displayed a better protective effect against dextran sulfate sodium- (DSS-) induced UC compared with 5-aminosalicylic acid (5-ASA) in C57BL/6 mice. The enhanced Nrf2 signaling and activated AKT/ERK were also observed in the colon of mice after SSM administration. These results first demonstrate the protective effect of SSM against UC and indicate that the effect is associated with AKT/ERK activation and subsequent Nrf2 signaling enhancement. This study provides a new insight into the medicinal value of SSM and proposes it as a new natural nutrition for better managing the symptoms of UC.

The therapeutic effect of Ilex pubescens extract on blood stasis model rats according to serum metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilex pubescens Hook. et Arn (MDQ), a traditional Chinese herb, is used in the treatment of cardiovascular diseases. However, the mechanisms underlying the preventive effect of MDQ on blood stasis remain unclear. AIM OF THE STUDY: In this study, serum metabolomics integrated with a biochemical assay strategy were established to evaluate the preventive effect and mechanism of action of MDQ on rats with acute blood stasis. MATERIALS AND METHODS: Forty-nine rats were divided into seven groups: the control group, model group, aspirin treatment group (30 mg/kg), clopidogrel treatment group (8 mg/kg) and three MDQ treatment groups (250, 500 and 1000 mg/kg). A hybrid quadrupole time of flight mass spectrometry (QTOF/MS) coupled to ultra-high-performance liquid chromatography (UPLC) was applied for profiling the serum metabolites. The multivariate data analysis techniques using unsupervised principal component analysis (PCA) and supervised orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used for pattern recognition and distinguishing variabilities among groups. RESULTS: MDQ protected the rats against blood stasis, as evidenced by the restoration of the anti-platelet aggregation activity, fibrinogen concentration, prothrombin time, thrombin time, activated partial thromboplastin time, endothelial nitric oxide synthase, endothelin, thromboxane B2 and 6-keto-prostaglandin F1α. The combination of PCA and OPLS-DA revealed deviations in eighteen differential biomarkers in serum. The identified biomarkers were primarily engaged in the metabolic pathways including arachidonic acid metabolism, glycerophospholipid metabolism, phospholipid biosynthesis and bile acid biosynthesis. The levels of eleven biomarkers showed significant alterations and a tendency to be restored to normal values in MDQ-treated blood stasis rats. Moreover, a correlation network diagram was constructed to show the serum biomarkers perturbed by MDQ. CONCLUSIONS: These results suggested that MDQ had preventive effects on blood stasis in rats via arachidonic acid metabolism and glycerophospholipid metabolism.

Polymorphisms of peroxisome proliferator-activated receptor γ (PPARγ) and cluster of differentiation 36 (CD36) associated with valproate-induced obesity in epileptic patients.

RATIONALE: Valproate (VPA) is a choice for the treatment of primary generalized epilepsies and partial epilepsies. Unfortunately, weight gain or obesity is one of the most frequent adverse effects of VPA treatment. Genetic factors were shown to be involved in the effect. OBJECTIVE: The aim of this study was to investigate the association of selected single nucleotide polymorphisms (SNPs) of cluster of differentiation 36 (CD36) and peroxisome proliferator-activated receptor γ (PPARγ) with VPA-induced weight gain and obesity in epileptic patients. METHODS: A total of 225 Chinese Han epilepsy patients receiving VPA treatment were recruited in the study. Height and weight for the calculation of body mass index (BMI) were measured at the initiation of VPA therapy and in the follow-up examination. A BMI of 25 kg/m2 or higher was defined as obesity on the basis of the World Health Organization (WHO) criteria for Asian populations. Four SNPs in CD36 (rs1194197, rs7807607) and PPARγ (rs10865710, rs2920502) were genotyped using the Sequenom® MassArray iPlex platform. RESULTS: About 19.6% of epileptic patients receiving VPA therapy were found to become obese. After covariate analysis of age, gender, sex, height, initial BMI, and VPA dosage, the CD36 rs1194197 C allele and rs7807607 T allele (OR, 0.31; 95%CI, 0.13-0.72; P = 0.009 and OR, 0.38; 95%CI; 0.18-0.83; P = 0.02, respectively) were identified as protective factors for VPA-induced obesity. The PPARγ rs10865710 C allele carriers were found to be less likely to suffer from VPA-induced obesity compared with GG genotype carriers (OR, 0.04; 95%CI, 0.01-0.12; P < 0.001). After a Bonferroni correction for multiple comparisons, the genotypic associations of CD36 rs1194197 and PPARγ rs10865710 and the allelic association of CD36 rs7807607 with obesity remained statistically significant. CONCLUSIONS: Our data first indicated that CD36 and PPARγ polymorphisms may be associated with VPA-induced obesity and weight gain, suggesting that CD36 and PPARγ may have potential value in predicting VPA-induced obesity in Chinese Han epileptic patients.

Synergistic immunoreaction of acupuncture-like dissolving microneedles containing thymopentin at acupoints in immune-suppressed rats.

Dissolving microneedles carried drug molecules can effectively penetrate the stratum corneum of skin to improve the transdermal drug delivery. The traditional Chinese medicine acupuncture is based on the needle stimulation at a specific location (acupoint) to generate and transmit biochemical and physiological signals which alter the pathophysiological state of patients. However, the pain associated with conventional acupuncture needles and the requirement of highly trained professionals limit the development of acupuncture in non-Asian countries. The purpose of this study is to investigate whether the dissolving microneedles can be utilized as a self-administered painless replacement for acupuncture and locally released drug molecules can achieve expected therapeutic outcomes. Immunosuppressive rats were treated with acupuncture at Zusanli (ST36) acupoint using microneedles containing thymopentin. The immune functions and psychological mood of the immunosuppressed animals were examined. The proliferation of splenocytes was examined by CCK-8 assay. CD4 and CD8 expression patterns in spleen cells were detected by flow cytometry. The current study showed that use of either microneedles containing thymopentin or conventional acupuncture both resulted in immune cell proliferation, which was confirmed by flow cytometry. Furthermore, either conventional acupuncture or microneedles were able to effectively mitigate the anxiety caused by immune-suppression when applied on the ST36.

Simultaneous determination of trimethylamine N-oxide, choline, betaine by UPLC-MS/MS in human plasma: An application in acute stroke patients.

Trimethylamine-N-oxide (TMAO) is derived from the gut microbiome and tissues metabolism of dietary choline and betaine. These molecules are closely related to the development of cardiovascular and cerebrovascular diseases. A rapid, sensitive and accurate method has been developed and validated for the simultaneous determination of trimethylamine N-oxide (TMAO), choline and betaine in human plasma using d9-trimethylamine N-oxide (TMAO), d9-choline, d9-betaine as the internal standard (IS). After methanol precipitation with 10 µL plasma samples, the analytes were extracted and then separated on Amide column (2.1 × 100 mm, 1.7 µm, waters) with an isocratic elution program consisting of acetonitrile-water (containing 10 mM ammonium formate pH = 3.0) at a flow of 400 µL/min. The detection was achieved under the selected reaction monitoring (SRM) scan using positive electrospray ionization (ESI+) in 3 min. The mass transitions monitored were as follows: m/z 76.3 → 58.4 for TMAO, m/z 104.2 → 60.3 for choline, m/z 118.1 → 58.3 for betaine, m/z 85.1 → 66.3 for d9-TMAO, m/z 113.2 → 69.3 for d9-choline, and m/z 127.1 → 67.2 for d9-betaine, respectively. The method has been fully validated for specificity, lower limit of quantification, linearity, stability, intra- and inter-day accuracy and precision. This assay combines simple sample processing with a short run time and small plasma volumes, making it well suited for high-throughput routine clinical or research purposes. The newly developed method was successfully applied to the patients (n = 220) suffered from acute stroke, and the concentration of choline was firstly found to be closely related with the prognosis of these patients.

Inhibition of Glucose-6-Phosphate Dehydrogenase Reverses Cisplatin Resistance in Lung Cancer Cells via the Redox System.

The pentose phosphate pathway (PPP), which branches from glycolysis, is correlated with cancer cell proliferation, survival and senescence. In this study, differences in the metabolic profile of the PPP and the redox status of human lung carcinoma A549 cells and cisplatin-induced multidrug-resistant A549/DDP cells were analyzed and evaluated. The results showed that A549/DDP cells exhibited differential PPP-derived metabolic features and redox-related molecules. A549/DDP cells exhibited increased expression and enzymatic activity of PPP enzyme glucose-6-phosphate dehydrogenase (G6PD). Furthermore, as demonstrated by the apoptotic rate, cell viability, and colony formation, inhibition of G6PD by siRNA or an inhibitor sensitized A549/DDP cells to cisplatin. Additionally, inhibition of G6PD restored the cisplatin sensitivity of A549/DDP cells by influencing redox homeostasis. In conclusion, overcoming cisplatin resistance through inhibition of G6PD could improve the understanding of the mechanisms underlying cisplatin-induced resistance in human lung cancer and may provide insights into the therapeutic potential of this treatment to combat resistance.

Valproate induced hepatic steatosis by enhanced fatty acid uptake and triglyceride synthesis.

Steatosis is the characteristic type of VPA-induced hepatotoxicity and may result in life-threatening hepatic lesion. Approximately 61% of patients treated with VPA have been diagnosed with hepatic steatosis through ultrasound examination. However, the mechanisms underlying VPA-induced intracellular fat accumulation are not yet fully understood. Here we demonstrated the involvement of fatty acid uptake and lipogenesis in VPA-induced hepatic steatosis in vitro and in vivo by using quantitative real-time PCR (qRT-PCR) analysis, western blotting analysis, fatty acid uptake assays, Nile Red staining assays, and Oil Red O staining assays. Specifically, we found that the expression of cluster of differentiation 36 (CD36), an important fatty acid transport, and diacylglycerol acyltransferase 2 (DGAT2) were significantly up-regulated in HepG2 cells and livers of C57B/6J mice after treatment with VPA. Furthermore, VPA treatment remarkably enhanced the efficiency of fatty acid uptake mediated by CD36, while this effect was abolished by the interference with CD36-specific siRNA. Also, VPA treatment significantly increased DGAT2 expression as a result of the inhibition of mitogen-activated protein kinase kinase (MEK) - extracellular regulated kinase (ERK) pathway; however, DGAT2 knockdown significantly alleviated VPA-induced intracellular lipid accumulation. Additionally, we also found that sterol regulatory element binding protein-1c (SREBP-1c)-mediated fatty acid synthesis may be not involved in VPA-induced hepatic steatosis. Overall, VPA-triggered over-regulation of CD36 and DGAT2 could be helpful for a better understanding of the mechanisms underlying VPA-induced hepatic steatosis and may offer novel therapeutic strategies to combat VPA-induced hepatotoxicity.