Natural pentacyclic triterpenoid from Pristimerin sensitizes p53-deficient tumor to PARP inhibitor by ubiquitination of Chk1.

Inhibition of checkpoint kinase 1 (Chk1) has shown to overcome resistance to poly (ADP-ribose) polymerase (PARP) inhibitors and expand the clinical utility of PARP inhibitors in a broad range of human cancers. Pristimerin, a naturally occurring pentacyclic triterpenoid, has been the focus of intensive studies for its anticancer potential. However, it is not yet known whether low dose of pristimerin can be combined with PARP inhibitors by targeting Chk1 signaling pathway. In this study, we investigated the efficacy, safety and molecular mechanisms of the synergistic effect produced by the combination olaparib and pristimerin in TP53-deficient and BRCA-proficient cell models. As a result, an increased expression of Chk1 was correlated with TP53 mutation, and pristimerin preferentially sensitized p53-defective cells to olaparib. The combination of olaparib and pristimerin resulted in a more pronounced abrogation of DNA synthesis and induction of DNA double-strand breaks (DSBs). Moreover, pristimerin disrupted the constitutional levels of Chk1 and DSB repair activities. Mechanistically, pristimerin promoted K48-linked polyubiquitination and proteasomal degradation of Chk1 while not affecting its kinase domain and activity. Importantly, combinatorial therapy led to a higher rate of tumor growth inhibition without apparent hematological toxicities. In addition, pristimerin suppressed olaparib-induced upregulation of Chk1 and enhanced olaparib-induced DSB marker γΗ2ΑΧ in vivo. Taken together, inhibition of Chk1 by pristimerin has been observed to induce DNA repair deficiency, which may expand the application of olaparib in BRCA-proficient cancers harboring TP53 mutations. Thus, pristimerin can be combined for PARP inhibitor-based therapy.

Animal models of brain and spinal cord metastases of NSCLC established using a brain stereotactic instrument.

Objective: Animal models of brain and spinal cord metastases of non-small cell lung cancer were established through the intracranial injection of PC-9 Luc cells with a brain stereotaxic device. This method provides a reliable modeling method for studying brain and spinal cord metastases of non-small cell lung cancer. Methods: PC-9 Luc cells at logarithmic growth stage were injected into the skulls of 5-week-old BALB/c nude mice at different cell volumes (30 × 104, 80 × 104) and different locations (using anterior fontanel as a location point, 1 mm from the coronal suture, and 1.5 mm from the sagittal suture on the right upper and right lower side of the skull). After 1 week of cell inoculation, fluorescence signals of tumor cells in the brain and spinal were detected using the IVIS Xenogen Imaging system. After 4 weeks, brain and spinal tissues from the nude mice were harvested. Following paraffin-embedded sectioning, HE staining was performed on the tissues. Results: The fluorescence signals revealed that both brain and spinal cord metastasis occurred in the mice where the cells were injected at the lower right side of the skull. There was only brain metastasis in the nude mice injected with 30 × 104 cells at the upper right side of the skull. Both brain and spinal cord metastasis occurred in the nude mice injected with 80 × 104 cells. The HE staining revealed that both brain and spinal cord metastasis occurred in the mice injected with different amounts of PC-9 Luc cells, consistent with the results detected using the IVIS Xenogen Imaging system, thereby demonstrating the reliability of detecting fluorescent signals in vivo to determine tumor growth. Conclusion: It is a reliable method to establish the animal model of brain and spinal cord metastases of non-small cell lung cancer by injecting different quantities of cells from different positions with a brain stereotaxic device. The IVIS Xenogen Imaging system has high reliability in detecting the fluorescence signals of brain and spinal cord metastatic tumors.

Downregulation of CLDN6 inhibits cell migration and invasion and promotes apoptosis by regulation of the JAK2/STAT3 signaling pathway in hepatocellular carcinoma.

Background: High expression of CLDN6 in hepatocellular carcinoma (HCC) has been widely reported. During this research, CLDN6's effect on the infiltration, migration, and apoptosis of HCC cells was investigated. Methods: Initially, the knockdown and overexpression of CLDN6 in HCC cells were carried out by short interfering RNA (siRNA) and plasmid transfection. The transfection efficiency was detected by means of a quantitative real-time polymerase chain reaction (qRT-PCR) assay, immunofluorescence staining, and Western blot analysis. Transwell and wound-healing assays were employed for the detection of invasion and migration ability. CCK-8 assay and flow cytometry were utilized for the detection of apoptosis. Finally, analysis of the expression of pathway-related proteins (JAK2, STAT3, p-JAK2, and p-STAT3) and the regulation of apoptotic responses (by measurement of cleaved caspase-3, Bax, and Bcl-2 levels) was carried out. Results: When CLDN6 was knocked down, the cellular invasion and migration ability decreased, and apoptosis increased, which decreased p-JAK2, p-STAT3, and anti-apoptotic protein bcl-2 expression. Furthermore, an elevation was observed in cleaved caspase-3 and Bax expression levels. Contrarily, upon overexpression of CDLN6, the aforementioned experimental results were reversed. Conclusions: CLDN6 knockdown results in the inhibition of HCC cells' infiltration and migration and promotes apoptosis via downregulation of the JAK2/STAT3 signaling pathway.

Sodium pyruvate exerts protective effects against cigarette smoke extract-induced ferroptosis in alveolar and bronchial epithelial cells through the GPX4/Nrf2 axis.

BACKGROUND: Ferroptosis in alveolar and bronchial epithelial cells is one of the main mechanisms underlying the development of chronic obstructive pulmonary disease (COPD). Sodium pyruvate (NaPyr) is a natural antioxidant in the body, exhibiting anti-inflammatory and antioxidant activities. NaPyr has been used in a Phase II clinical trial as a novel therapy for COPD; however, the mechanism underlying NaPyr-mediated therapeutic benefits in COPD is not well understood. OBJECTIVE: We aimed to assess the protective effects of NaPyr and elucidate its potential mechanism in cigarette smoke extract (CSE)-induced ferroptosis.To minic the inflammatory response and ferroptosis triggered by cigarette smoke in COPD in an invitro cell based system, we expose a human bronchial epithelial cells to CSE. METHODS: To minic the inflammatory response and ferroptosis triggered by cigarette smoke in COPD in an invitro cell based system, the A549 (human lung carcinoma epithelial cells) and BEAS-2B (bronchial epithelial cells) cell lines were cultured, followed by treatment with CSE. To measure cellular viability and iron levels, we determined the levels of malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), mitochondrial superoxide (MitoSOX), membrane potential (MMP), and inflammatory factors (tumor necrosis factor [TNF] and interleukin [IL]-8), and examined CSE-induced pulmonary inflammation and ferroptosis. To clarify the molecular mechanisms of NaPyr in COPD therapy, we performed western blotting and real-time PCR (qPCR) to determine the expression of glutathione peroxidase 4 (GPX4), nuclear factor E2-related factor 2 (Nrf2), and cyclooxygenase 2 (COX2). RESULTS: We found that NaPyr effectively mitigated CSE-induced apoptosis and improved apoptosis induced by erastin, a ferroptosis inducer. NaPyr significantly decreased iron and MDA levels and increased GSH levels in CSE-induced cells. Furthermore, NaPyr suppressed ferroptosis characteristics, such as decreased levels of ROS, MitoSOX, and MMP. NaPyr significantly increases the expression levels of GPX4 and Nrf2, indicating that activation of the GPX4/Nrf2 axis could inhibit ferroptosis in alveolar and bronchial epithelial cells. More importantly, NaPyr inhibited the secretion of downstream inflammatory factors, including TNF and IL-8, by decreasing COX2 expression levels to suppress CSE-induced inflammation. CONCLUSION: Accordingly, NaPyr could mitigate CSE-induced ferroptosis in alveolar and bronchial epithelial cells by activating the GPX4/Nrf2 axis and decreasing COX2 expression levels. In addition, NaPyr reduced the secretion of inflammatory factors (TNF and IL-8), affording a novel therapeutic candidate for COPD.

MPZL1 Promotes Lung Adenocarcinoma Progression by Enhancing Tumor Proliferation, Invasion, Migration, and Suppressing Immune Function via Transforming Growth Factor-ß1.

Lung cancer (LC) is the leading cause of death worldwide, and lung adenocarcinoma (LUAD) is the most common form of LC. The abnormally high expression of myelin protein zero-like 1 (MPZL1) promotes the malignant progression of various tumors. However, there is no relevant report on the functional role of MPZL1 in LUAU. In this study, we applied Illumina sequencing to screen differentially expressed genes. Subsequently, MPZL1 was selected as hub gene for quantitative real-time polymerase chain reaction (qRT-PCR) and CCK8 assay. The expression level of MPZL1 was analyzed by immunohistochemistry, immunofluorescence, western blot, and qRT-PCR. After silencing or overexpressing MPZL1, CCK8, EDU, clone formation, scratch healing, invasion, and nude mouse tumor-bearing experiments were performed to detect the abilities of cell proliferation, migration, invasion, and tumorigenicity. Moreover, qRT-PCR, western blot, coimmunoprecipitation, and scratch healing assays were conducted to explore the transcriptional regulatory factors of MPZL1. Finally, the relationship between MPZL1 and immunotherapy was explored through public databases and validated in vivo. The results show that a total of 196 high-expressed genes and 496 low-expressed genes were screened. Differential genes are mainly enriched in cell proliferation and division, protein binding, and other pathways and functions. MPZL1 was selected as the hub gene and upregulated in LUAD tissues and cells. Silencing MPZL1 inhibited the cell proliferation and cloning formation, and the growth of tumor. Conversely, overexpression of MPZL1 has the opposite effect. In addition, MPZL1 combines with the transforming growth factor-ß1 to promote the progress of LUAD. Finally, we found that high expression of MPZL1 is negatively correlated with infiltration of CD8+ cells and may lead to immunotherapy resistance. In summary, this study revealed a new mechanism by which MPZL1 promotes LUAD progression by enhancing tumor proliferation, invasion, migration, and suppressing immune function.

Experimental study of EGFR-TKI aumolertinib combined with ionizing radiation in EGFR mutated NSCLC brain metastases tumor.

Aumolertinib is an irreversible third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), although it has been administered for the treatment of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). However, it is unclear whether aumolertinib combined with ionizing radiation (IR) has potential therapeutic effects in treating brain metastases (BM) tumors from NSCLC. This study explored the anti-tumor effects of aumolertinib combined with IR in epidermal growth factor receptor mutated (EGFRm) NSCLC BM tumors. First, we established a xenograft model of NSCLC BM tumors in BALB/c nude mice and assessed the anti-tumor effects of this combination. Furthermore, we examined the concentrations of aumolertinib in brain tissue and blood using liquid chromatography-mass spectrometry (LC-MS); after that, we used CCK-8, colony formation, flow cytometry assay, and immunofluorescence staining to detect the effects of aumolertinib combined with IR upon PC-9 and NCI-H1975 cells, such as cell proliferation, survival, apoptosis, cycle distribution, the situation of DNA damage, and the expression levels of relevant proteins which were detected via western blotting; finally, we chose a clinical case with which to explore the clinical benefits to the EGFRm NSCLC BM patient after the treatment of the aforementioned combination. The experiments of NSCLC BM tumor animal models demonstrated that the combination enhanced the therapeutic effects and increased the intracranial accumulation of aumolertinib; the combination can inhibit cell proliferation and survival, delay the repair of DNA damage, and increase the rates of cell apoptosis and aumolertinib abrogated G2/M phase arrest, which the IR induced; the clinical study verified that the combination demonstrated better patient benefits. In conclusion, our study demonstrated that combining aumolertinib and IR has promising anti-tumor effects in EGFR-mutant NSCLC and that this combined treatment modality may be employed as a potential therapeutic strategy for EGFR-mutant NSCLC BM patients clinically.

Salvianolic acid B, a new type I IRE1 kinase inhibitor, abrogates AngII-induced angiogenesis by interacting with IRE1 in its active conformation.

Angiotensin II (AngII)-mediated pathological angiogenesis is one of the important factors promoting the progression of atherosclerosis, tumour metastasis, and diabetic retinopathy. Here, we first demonstrate that salvianolic acid B (Sal B) attenuated AngII-induced angiogenesis by downregulating the IRE1/ASK1/JNK/p38MAPK signalling pathway and protected vascular endothelial cells from hypoxia-induced damage. These pharmacological consequences could be ascribed to the unique interactions between Sal B and the ATP-binding cavity of IREIα, leading to bi-directional roles of IRE1 kinase and endonuclease activity; this may possibly be one of the essential mechanisms of the bi-directional regulation of angiogenesis in different conditions. Moreover, our results indicated that IRE1 was a novel anti-angiogenesis target and type I IRE1 kinase inhibitor (e.g., Sal B, APY29) and might be a potentially eligible low-toxicity drug for treating AngII-mediated pathological angiogenesis.

Baicalein-ameliorated cerebral ischemia-reperfusion injury dependent on calpain 1/AIF pathway.

Cerebral ischemia reperfusion (CIR) has become the leading cause of death and disability. Baicalein is a natural bioactive ingredient extracted from Scutellaria baicalensis Georgi and has neuroprotective activity. In our work, baicalein was found to reduce neurological deficits, brain water content, infarct area, and neuronal death of rats induced by middle cerebral artery occlusion/reperfusion. In vitro, oxygen-glucose deprivation/reperfusion induced inordinate ROS production and apoptosis that could be reversed by baicalein. Our study revealed for the first time that baicalein has the potential to bind and inhibit the activity of calpain 1, thereby inhibiting AIF nuclear translocation. These findings demonstrated that baicalein protected against CIR injury via inhibiting AIF nuclear translocation by inhibiting calpain 1 activity.

Effect of almonertinib on the proliferation, invasion, and migration in non-small cell lung cancer cells. / 阿美替尼对非小细胞肺癌细胞增殖、侵袭和迁移的影响.

OBJECTIVES: Lung cancer is one of the most common malignant tumors in the world, and its lethality ranks the first among many malignant tumors. For non-small cell lung cancer (NSCLC) patients, due to the high mortality rate, the overall 5-year survival rate is less than 15%. When NSCLC undergoes local invasion, the 5-year survival rate is only 20%, and it is even lower when distant metastasis occurs up to 4%. Almonertinib is an innovative drug independently researched and developed by China with independent intellectual property rights. As an epidermal growth factor receptor tyrosine kinase inhibitor, almonertinib is mainly used for locally advanced or metastatic NSCLC patients with epidermal growth factor receptor (EGFR) T790M mutation. This study aims to investigate the effects of almonertinib on the proliferation, invasion and migration of NSCLC cells in vitro. METHODS: NSCLC cells H1975 and PC-9 were cultured in vitro. The effects of almonertinib on the proliferation, apoptosis, invasion, and migration of H1975 and PC-9 cells were detected by CCK-8 assay, apoptotic assay and Transwell assay. The expression of invasion and migration related proteins was detected by Western blotting. RESULTS: The CCK-8 experiment showed that almonertinib inhibited the proliferation of H1975 and PC-9 cells in a time- and dose-dependent manner. The IC50 values in PC-9 cells at 24 and 48 h were 5.422 and 1.302 µmol/L, respectively. The IC50 values in H1975 cells at 24 and 48 h were 4.803 and 2.094 µmol/L, respectively. Almonertinib (1, 2, 4, 8 µmol/L)-treated PC-9 and H1975 cells for 24 h resulted in apoptosis rate at (8.82±3.22)%, (9.53±4.24)%, (13.62±3.69)%, (42.10±1.76)% and (9.81±0.90)%, (10.51±1.49)%, (15.34±3.50)%, (28.97±2.57)%, respectively. The transwell experiment showed that almonertinib inhibited the invasion and migration of H1975 and PC-9 cells. Western blotting showed that compared with the control group, the expression levels of MMP-9, MMP-2 and vimentin protein in PC-9 and H1975 cells in 1, 2 and 4 µmol/L almonertinib treatment group were significantly lower, and the expression level of E-cadherin protein was significantly higher (all P<0.05). The experimental results of nude mice showed that compared with the control group and the positive control ositinib (AZD9291) group, the tumor growth was significantly inhibited, the weight of nude mice, the tumor volume and the tumor mass were significantly reduced in the almonertinib treatment group (all P<0.05). CONCLUSIONS: Almonertinib can inhibit the proliferation, invasion, and migration of NSCLCH1975 and PC-9 cells in vitro and vivo, and promote the apoptosis of H1975 and PC-9 cells. The underlying mechanism may be related to the inhibition of tumor cell epithelial mesenchymal transformation and metalloproteinase expression.

Experimental Study of Almonertinib Crossing the Blood-Brain Barrier in EGFR-Mutant NSCLC Brain Metastasis and Spinal Cord Metastasis Models.

Roughly one third of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI)-sensitive mutated (EGFRm) tumors experience disease progression through central nervous system (CNS) metastases during treatment. Although EGFR-TKIs have been reported to be favored in some patients with EGFRm NSCLC CNS metastases, novel EGFR-TKIs with proven efficacy in CNS pathologies are clinically needed.To investigate whether almonertinib, a novel third-generation EGFR-TKI for NSCLC, can cross the blood-brain barrier (BBB) and deliver treatment for EGFR-mutant NSCLC brain metastases and spinal cord metastases, we constructed NSCLC brain metastasis and spinal cord metastasis models in vivo to observe the anti-tumor effects of almonertinib. Using ABCB1-MDCK and BCRP-MDCK monolayer cells as the in vitro study model, the effects of transport time and drug concentration on the apparent permeability coefficient of almonertinib and its active metabolite, HAS-719, were investigated. The results of this study show that almonertinib can significantly inhibit PC9 brain and spinal cord metastases. Pharmacokinetic studies in mice revealed that almonertinib has good BBB penetration ability, whereas the metabolite HAS-719 does not easily penetrate the BBB. Early clinical evidence of almonertinib activity in patients with EGFRm-advanced NSCLC and brain metastases has also been reported. In conclusion, almonertinib easily penetrates the BBB and inhibits advanced NSCLC brain and spinal cord metastases.

Bullatacin triggers immunogenic cell death of colon cancer cells by activating endoplasmic reticulum chaperones.

BACKGROUND: It is well accepted that the immune system efficiently contributes to positive outcomes of chemotherapeutic cancer treatment by activating immunogenic cell death (ICD). However, only a limited number of ICD-inducing compounds are well characterized at present; therefore, identification of novel ICD inducers is urgently needed for cancer drug discovery, and the need is becoming increasingly urgent. METHODS: Herein, we assessed the antitumour activity of bullatacin by MTS assay and apoptosis assay. ICD biomarkers, such as calreticulin (CRT), high-mobility group protein B1 (HMGB-1), heat shock protein (HSP)70, HSP90 and ATP, were assessed by Western blotting, ELISA and flow cytometry. Western blot and qPCR assays were performed to explore the underlying mechanisms of bullatacin-induced ICD. Flow cytometry was used to detect macrophage phagocytosis. RESULTS: First, bullatacin induced apoptosis in both SW480 cells and HT-29 cells in a time-dependent manner at 10 nM, as assessed by flow cytometry. Moreover, Western blot and flow cytometry assays showed that CRT and HSP90 (biomarkers of early ICD) significantly accumulated on the cell membrane surface after approximately 6 h of treatment with bullatacin. In addition, ELISAs and Western blot assays showed that the second set of hallmarks required for ICD (HMGB1, HSP70 and HSP90) were released in the conditioned media of both SW480 and HT-29 cells after 36 h of treatment. Furthermore, qPCR and Western blot assays indicated that bullatacin triggered ICD via activation of the endoplasmic reticulum stress (ERS) signalling pathway. Finally, bullatacin promoted macrophage phagocytosis. CONCLUSION: This study documents that bullatacin, a novel ICD inducer, triggers immunogenic tumour cell death by activating ERS even at a relatively low concentration in vitro.

Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib.

The emergence of secondary resistance is the main failure cause of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) as a targeted therapy for non-small cell lung cancer (NSCLC). EGFR mutations of NSCLC cells can markedly increase glutamine transporter (SLC1A5) expression, thereby increasing glutamine metabolism. Glutamine metabolites can activate EGFR downstream signals, including mTOR, ERK1/2, STAT3, etc., which is an important cause for the decreased sensitivity of NSCLC to EGFR-TKIs. CCK8 and Annexin V/PI assays were conducted to detect the effects of Almonertinib and/or V9302 on the proliferation and apoptosis of NSCLC cells. Proteomics was used to determine the effect of Almonertinib on energy metabolism-related proteins in NSCLC. siRNA transfection was performed to study the effect of SLC1A5 down-regulation on cell proliferation. In addition, the effects of drugs on colony formation capacity were determined by colony formation assay. Immunofluorescence and Western blot were utilized to detect the apoptosis- and autophagy-related proteins expression. DAPI staining was utilized to detect the effect of drugs on the nucleus. Transmission electron microscope was used to observe the changes of submicroscopic structure such as autophagosomes and nucleus of cells. mCherry-GFP-LC3B tandem fluorescent protein was to used to detect the level of autophagy flux. Tumor-bearing nude mouse model was utilized to detect the effect of V9302 on the anti-tumor effect of Almonertinib in vivo. As a result, Almonertinib suppressed H1975 and A549 cell proliferation depended on its dosage and treatment duration, and it also induced apoptosis. A549 cells with wild-type EGFR had lower sensitivity to Almonertinib. The expression of SLC1A5 was up-regulated by stimulating with low concentration of Almonertinib in NSCLC cells. SLC1A5 was highly expressed in A549 cells with wild-type EGFR. Glutamine deletion or SLC1A5 inhibition/silencing inhibited the proliferation of NSCLC cells, and decreased cellular glutamine uptake. The combination of SLC1A5 inhibitor V9302 and Almonertinib had a synergistic inhibitory effect on the proliferation of NSCLC. V9302 enhanced the effect of Almonertinib in apoptosis-inducing in NSCLC cells. The combination of V9302 and Almonertinib might induce apoptosis by inhibiting autophagy.

[EGFR tyrosine kinase inhibitor HS-10296 induces autophagy and apoptosis in triplenegative breast cancer MDA-MB-231 cells].

OBJECTIVE: To investigate the inhibitory effect of epidermal growth factor receptor tyrosine kinase inhibitor (EGFRTKI) HS-10296 on the proliferation of triple-negative breast cancer (TNBC) MDA-MB-231 cells and explore the possible molecular mechanism. METHODS: MDA-MB-231 cells were treated with HS-10296 for 24, 48, or 72 h, and CCK-8 assay was used to assess the changes in the cell viability. The inhibitory effect of HS-10296 on cell proliferation was determined by clonogenic assay. JC-1 and flow cytometry were employed for analyzing the cell apoptosis, and the ultrastructure of the cells was observed under electron microscope. After pretreatment with autophagy inhibitor chloroquine (CQ), MDA-MB-231 cells were divided into control group, CQ treatment group, HS-10296 (4 and 6 µmol/L) treatment groups and combined treatment groups, and the sensitivity of the treated cells to HS-10296 was determined using CCK-8 assay. The effects of HS-10296 on EGFR pathway and apoptosis- and autophagy-related proteins in MDA-MB-231 cells were investigated using Western blotting. RESULTS: HS-10296 significantly inhibited the proliferation of MDA-MB-231 cells with IC50 values at 24, 48 and 72 h of 8.393, 2.777 and 2.016 µmol/L, respectively. JC-1 and flow cytometry showed that HS-10296 induced obvious apoptosis of MDA-MB-231 cells, which showed an apoptosis rate of (21.63 ± 2.97)% following treatment with 8 µmol/L HS-10296. Autophagy vesicles were observed in the cells treated with HS-10296 under electron microscope. In MDA-MB-231 cells pretreated with CQ, inhibition of autophagy significantly enhanced HS-10296-induced cell death. Western blotting showed that the apoptosis-related protein caspase-3 was activated after HS-10296 treatment to cut its substrate PARP. The expression of autophagy-related protein light chain 3B (LC3B) was significantly enhanced after HS-10296 treatment (P < 0.01), which also resulted in inhibited phosphorylation of EGFR and AKT proteins in the cells. CONCLUSIONS: HS-10296 can inhibit the proliferation and induce autophagy and apoptosis of MDA-MB-231 cells by inhibiting the EGFR/PI3K/AKT signaling pathway.

[Gefitinib inhibits glycolysis and induces programmed cell death in non-small cell lung cancer cells].

OBJECTIVE: To observe the cell death pattern induced by gefitinib in non-small cell lung cancer A549 and H1975 cells and explore the possible mechanism in light of glycolysis. METHODS: The inhibitory effects of gefitinib at 20, 30, or 40 µmol/L in A549 cells and at 20, 40, or 80 µmol/L in H1975 cells were examined using MTT assay. The changes of lactic acid level in the cells were determined with a lactic acid kit, and the expression levels of glycolysis-related proteins (PKM2 and HK2) and the proteins in PI3K-Akt-mTOR signaling pathway were detected using Western blotting. 2-NBDG was used for detecting glucose uptake capacity of the cells, and ATP kit was used to detect the intracellular ATP level. The mitochondrial membrane potential of the cells was examined with the JC-1 kit, and cell apoptosis was analyzed with Annexin V-FITC/PI double staining. The relative expression levels of the apoptotic proteins Bax and Bcl-2 and the autophagy marker protein LC3B were detected with Western blotting. RESULTS: MTT assay showed that gefitinib inhibited the proliferation of A549 and H1975 cells in a time- and dose-dependent manner (P < 0.05). The IC50 of gefitinib at 24, 48 and 72 h was 48.6, 28.6 and 19.7 µmol/L in A549 cells and was 321.6, 49.1 and 14.6 µmol/L in H1975 cells, respectively. Gefitinib significantly lowered intracellular lactic acid level of the cells (P < 0.05) and down-regulated the expressions of PKM2 and HK2 proteins (P < 0.05) and PI3K-Akt-mTOR signaling pathway-associated proteins (P < 0.05). Gefitinib obviously inhibited glucose uptake and ATP levels in both A549 and H1975 cells (P < 0.05). Treatment with gefitinib induced obviously enhanced apoptosis in the cells, resulting in apoptosis rates of (10.77± 1.0)%, (14.5±0.4)%, (17.4±0.2)% and (32.1±0.6)% at 0, 20, 30 and 40 µmol/L in A549 cells (P < 0.05) and of (10.5±0.6)%, (13.2± 0.92)%, (18.9±0.98)% and (35.1±1.4)% at 0, 20, 40 and 80 µmol/L in H1975 cells, respectively (P < 0.05). The protein expression of Bax increased and that of Bcl-2 decreased following gefitinib treatment in the cells (P < 0.05). Gefitinib significantly increased autophagy in A549 and H1975 cells as shown by increased LC3B expressions following the treatment (P < 0.05). CONCLUSIONS: Gefitinib can inhibit the proliferation, induce apoptosis and increase autophagy in A549 and H1975 cells. Gefitinib induces apoptosis of the cells possibly by affecting glycolysis and PI3K-Akt-mTOR signaling pathway.

Different types of effective fractions from Radix Isatidis revealed a multiple-target synergy effect against respiratory syncytial virus through RIG-I and MDA5 signaling pathways, a pilot study to testify the theory of superposition of traditional Chinese Medicine efficacy.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Isatidis, a commonly used traditional Chinese medicine, is also documented in "Dictionary of Chinese Ethnic Medicine" being as an ethnic herb clinically utilized by different nations in China such as Mongol, Uygur, and Dong et al. It has been reported to have a very strong efficacy on respiratory viruses, but to date the mechanism remains unknown. Similarly, it is unclear how different types of effective fractions of Radix Isatidis interact to exert antiviral effects. AIM OF STUDY: To reveal the underlying mechanisms for the inhibitory effects of three active fractions from Radix Isatidis, i.e. total alkaloids, lignans and organic acids, on respiratory syncytial virus when used alone or in combination. In addition, we investigated whether these three parts worked synergistically in vivo and in vitro. MATERIALS AND METHODS: A mouse model of RSV infection was constructed by intranasal infection, and the pathological changes of lung tissues in different parts were observed. The level changes of IFNß and inflammatory cytokines in the mouse alveolar lavage fluid were detected by enzyme-linked immunosorbent assay (ELISA). The anti-RSV effects of different effective fractions were evaluated by the plaque reduction test. The mRNA and protein expressions of RIG-I, MDA-5, MAVS and IRF3 in RAW264.7 cells were detected by RT-PCR and Western blot respectively. RESULTS: HE staining showed that Radix Isatidis extracts alone or in combination relieved virus-induced mouse lung lesions. Compared with individual drugs, the lung lesions were alleviated more significantly after treatment with the three fractions in combination. ELISA demonstrated that the expression levels of IFNß and inflammatory cytokines were maintained balanced between antiviral and proinflammatory effects. The plaque reduction test indicated that the antiviral effect of combination treatment was much stronger than those of individual drugs. RT-qPCR and Western blot suggested that the mRNA and protein expression levels of key signaling molecules in the RIG-I and MDA5 pathways in mouse macrophages were down-regulated by different effective parts alone or in combination. CONCLUSIONS: The three effective fractions of Radix Isatidis have remarkable synergistic anti-RSV effects in vitro and in vivo, and total alkaloids and lignans show multi-target synergistic effects via the RIG-I and MDA5 signaling pathways.

The Anticancer Effects of Atractylenolide III Associate With the Downregulation of Jak3/Stat3-Dependent IDO Expression.

Objective: Indoleamin-2,3-dioxygenase-1 (IDO) has been identified as a checkpoint protein involved in generating the immunosuppressive tumor microenvironment that supports tumor growth. It has been reported that atractylenolide III (ATLIII) has anticancer and immune modulatory effects. This study is to determine the anticancer effects of ATLIII with the Jak3/Stat3-dependent IDO inactivation. Methods: We assessed the cytotoxicity of ATLIII and IFN-γ on lung cancer cells by MTT. We determined the efficacy of ATLIII on IFN-γ-induced IDO expression by RT-PCR and Western blot. We also determined the efficacy of ATLIII on Jak3/Stat3 pathway expression induced by IFN-γ and Jak3/Stat3-dependent IDO activation. Further molecular docking assay predicted the binding activity and site of ATLIII to Jak3 protein. Additional immunofluorescence staining was used to measure the Stat3 intracellular localization. Finally, we performed mouse animal experiments to observe changes in the expression of IDO, p-Jak3, p-Stat3, and tryptophan/kynurenine after ATLIII administration. Results: ATLIII showed no cytotoxicity at a wide of dosage range. ATLIII reduced the phosphorylation level of Jak3 and Stat3 in response to IFN-γ stimulation, then remarkably reduced the nuclear translocation of p-Stat3 by IFN-γ. Lastly, ATLIII significantly downregulated the expression level of IDO at a wide dosage range. Molecular docking assay showed that the oxygen atom on the five-membered ring of ATLIII was capable of forming a hydrogen bond with Leu905-NH2 site of Jak3 protein. Further evidence showed that though IFN-γ had normal capacity to trigger Stat3 phosphorylation, nuclear translocation, and promoter luciferase activity, ATLIII failed to trigger efficacy on reducing these changes under forced Jak3-Leu905 mutant expression condition. Finally, we confirmed this view in in vivo experiments. Conclusion: ATLIII has shown significant efficacy to inhibit IFN-γ-triggered Jak3/Stat3 pathway-dependent IDO activation, and do so through a direct binding to Jak3 protein. This study elucidated a new mechanism for the anticancer effect of ATLIII, which may provide a feasible target for the clinical immunotherapy of malignant tumors.

Resveratrol inhibits VEGF-induced angiogenesis in human endothelial cells associated with suppression of aerobic glycolysis via modulation of PKM2 nuclear translocation.

Endothelial cells (ECs) mainly depend on aerobic glycolysis to generate angiogenesis. Deregulation of glycolysis is often observed in human endothelial cells during angiogenesis. In the present study, we first report that resveratrol (RST), which has been intensively studied in glucose metabolism of various cancer cells, has a profound inhibitory effect on tube formation and migration via suppression of glycolysis in human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor (VEGF). Moreover, we further reveal that RST reduced the mRNA and protein level of glucose transporter-1(GLUT1), hexokinase II (HK2), phosphofructokinase-1(PFK1) and pyruvate kinase M2 (PKM2) through modulation of ERK-mediated PKM2 nuclear translocation. Our results provide a novel mechanism to account for the inhibition of RST on VEGF-mediated angiogenesis and suggest that targeting aerobic glycolysis or nuclear PKM2 may be a new approach for pathological angiogenesis prevention or treatment.

A spontaneous metastatic mathematical model in mice for screening anti-metastatic agents.

PURPOSE: A computational model based on clinical data from pancreatic cancer patients has been successfully created and used for predicting tumor sizes in primary and metastasis sites and survival time from kinetics of tumor cells, such as growth rate, metastasis rate and mutation rate, etc. Whether this computational model could be fitted or necessary modification of some parameters for fitting in mice is unknown. Here, we developed a computational model in mice for spontaneous metastasis to simulate the process of tumor metastasis based on the mathematical frameworks. METHODS: The spontaneous melanoma metastasis model in mice was used for assessing the fitting accuracy between the mathematical model and the experimental data and evaluating the efficacy of anticancer agents, as well as the invasion assay. RESULTS: According to the modified model, most of parameters including growth rate, mutation rate and metastasis rate, which were used to describe the whole metastatic course in mice were calculated based on the experimental analysis. Furthermore, only measurement of the growth rate of cancer in the primary site was required to predict the survival time. Our predicted results of the overall survival (OS) extension of mice were close to the clinical outcomes after treated with four clinical intervention strategies of CVD, Paclitaxel, Dartmouth and Temozolomide. And predictive efficacy of anticancer drug using the model matches well the factual experimental data in mice. CONCLUSIONS: The mathematical model is more economical and efficient for evaluating the tumor metastasis and could be used to screen the anti-cancer and anti-metastatic medicine by shortening the periods of assessment of OS extension in preclinical trials.

4(3H)-Quinazolone regulates innate immune signaling upon respiratory syncytial virus infection by moderately inhibiting the RIG-1 pathway in RAW264.7 cell.

The root of Isatis indigotica, also known as Ban-Lan-Gen (BLG), is commonly used for prevention and early treatment of respiratory virus infection, but the underlying mechanisms of action remain unclear. In the early stage of infection, the innate immune system is activated by virus, and related immune cells such as macrophages secrete large amounts of cytokines including IFNs and ILs. On the one hand, these cytokines can remove virus. On the other hand, they dominantly mediate the inflammatory injury caused by viral infection. This study evaluated the effects of the main active plasma components of BLG (tryptanthrin B, 4(3H)-Quinazolone and epigoitrin) on the innate immune response of respiratory syncytial virus (RSV)-activated mouse macrophages. ELISA, real-time PCR and reporter gene assay all showed that 4(3H)-Quinazolone inhibited RSV-induced IFN-ß secretion in mouse macrophages in a dose-dependent manner within a concentration range (0.3125-1.25µM) having no effects on cell viability, but the inhibitory effects were inferior to those of ribavirin. Western blot analyses further revealed that 4(3H)-Quinazolone inhibited RSV-induced expression of RIG-I (Retinoic acid-Inducible Gene-I) in mouse macrophages dose-dependently, thereby suppressing the transcription of IFN-ß, with lower effects than those of ribavirin. This may be one of the important mechanisms by which BLG inhibited inflammatory injury without affecting the immune system to eliminate virus. The results inspire future studies to elucidate the antiviral mechanisms of traditional Chinese medicine drugs.

Resveratrol suppresses pulmonary tumor metastasis by inhibiting platelet-mediated angiogenic responses.

BACKGROUND: To explore the impact of Resveratrol (RSV) on the angiogenic potential of activated platelets and to elucidate the underlying mechanism. METHODS: Vascular endothelial growth factor concentrations were measured by enzyme-linked immunosorbent assay. Capillary tube formation assay was used to examine the impact of RSV on the angiogenic potential of activated platelets. The levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate (cGMP) in the supernatant were evaluated using corresponding enzyme-linked immunosorbent assay kits. Immunoblotting assays were used to determine the expression of vasodilator-stimulated phosphoprotein and Akt phosphorylation. A pulmonary metastasis experiment with male nude mice model was performed to test the effect of RSV on pulmonary metastasis and angiogenesis in vivo. RESULTS: RSV inhibited platelets-mediated angiogenic responses induced by adenosine diphosphate (ADP)ADP through increased cGMP generation and cGMP-mediated vasodilator-stimulated phosphoprotein phosphorylation along with reduced intracellular Ca2+ mobilization. In addition, RSV attenuated the platelet secretion and angiogenic responses induced by A549 cells in vitro and suppressed A549 lung cancer metastasis and angiogenesis in nude mice. CONCLUSIONS: RSV is a potential therapeutic drug for the prevention of tumor metastasis by interrupting the platelet-tumor cell amplification loop.