Association of TOP2A and ADH1B with lipid levels and prognosis in patients with lung adenocarcinoma and squamous cell carcinoma.

BACKGROUND: Although lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) have different pathological and clinical features, they may share common driver genes. It was found that lipid levels can be used for early diagnosis of NSCLC; however, the relationship between driver genes and genes regulating lipid metabolism and their relationship with patient prognosis needs further investigation. METHODS: Genes whose expression was up- or down-regulated in both LUAD and LUSC were identified using the GEO database. Online tools like GEPIA 2, PrognoScan, UALCAN, and TIMER2.0 were used to investigate the association of these gene expressions with the patient's prognosis and lipid regulatory genes. The association between clinical lipid levels and the risk of LUAD and LUSC was analyzed by using a multiple logistic regression model. RESULTS: Topoisomerase II alpha (TOP2A) and alcohol dehydrogenase 1B (ADH1B) were identified as the only genes up- and down-regulated in both LUAD and LUSC. TOP2A and ADH1B expression levels significantly correlated with the patient's gender, age, individual cancer stage, histological subtype, nodal metastasis status, and TP53 mutation status. Additionally, only LUAD patients with higher TOP2A or lower ADH1B expressions displayed poor overall and relapse-free survival rates. Moreover, TOP2A levels exhibited a negative correlation with adipose triglyceride lipase (ATGL) and ATP-binding cassette transporter A1 (ABCA1) in both LUAD and LUSC. However, ADH1B showed inverse associations with the above-mentioned genes when compared to TOP2A expressions in both LUAD and LUSC. Furthermore, elevated triglyceride (OR = 1.59; 95% CI = 1.01 to 2.49; P < 0.05) and total cholesterol (OR = 2.45; 95% CI = 1.08 to 5.57; P < 0.05) levels might increase the risk of LUAD. CONCLUSIONS: TOP2A and ADH1B can be used as diagnostic markers for LUAD and LUSC, but only as independent prognostic factors for LUAD, and may be involved in lipid metabolism in LUAD patients but not in LUSC. Thus, combining genetic diagnostics with lipid panel tests might be an effective method for an early diagnosis and improved prognosis of LUAD.

OTUD4 regulates metastasis and chemoresistance in melanoma by stabilizing Snail1.

Melanoma is the most aggressive form of skin cancer with rapidly increased incidence worldwide especially in the Caucasian population. Surgical excision represents the curative treatment choice in patients with early-stage disease. However, the therapeutic outcomes in patients with metastatic melanoma remains unsatisfactory. Thus, understanding molecular mechanisms contributing to metastasis and chemoresistance is critical for new improved therapies of melanoma. Snail1, an important epithelial-mesenchymal transition transcription factors (EMT-TFs), is critical to induce the EMT process, thereby contributing to cancer metastasis. However, the involvement of Snail1 in melanoma metastasis remains elusive and the underlying mechanism to regulate Snail1 in melanoma needs to be further investigated. Here, we identified OTUD4 as a novel deubiquitinase of Snail1 in melanoma. Moreover, the depletion of OTUD4 in melanoma cells markedly inhibited Snail1 stability and Snail1-driven malignant phenotypes both in vitro and in vivo. Overall, our study establishes OTUD4 as a novel therapeutic target in metastasis and chemoresistance of melanoma by stabilizing Snail1 and provides a rationale for potential therapeutic strategies of melanoma.

Coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and epithelial-mesenchymal transition via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3ß signals.

Pneumoconiosis is the most common and serious disease among coal miners. In earlier work on this subject, we documented that coal dust (CD) nanoparticles (CD-NPs) induced pulmonary fibrosis (PF) more profoundly than did CD micron particles (CD-MPs), but the mechanism has not been thoroughly studied. Based on the GEO database, jveen, STRING, and Cytoscape tools were used to screen hub genes regulating PF. Particle size distribution of CD were analyzed with Malvern nanoparticle size potentiometer. Combining 8 computational methods, we found that IGF1, POSTN, MMP7, ASPN, and CXCL14 may act as hub genes regulating PF. Based on the high score of IGF1 and its important regulatory role in various tissue fibrosis, we selected it as the target gene in this study. Activation of the IGF1/IGF1R axis promoted CD-NPs-induced PF, and inhibition of the axis activation had the opposite effect in vitro and in vivo. Furthermore, activation of the IGF1/IGF1R axis induced generation of reactive oxygen species (ROS) to promote epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs) to accelerate PF. High-throughput gene sequencing based on lung tissue suggested that cytokine-cytokine receptor interaction and the NF-kB signaling pathway play a key role in PF. Also, ROS induced inflammation and EMT by the activation of the NF-kB/NLRP3 axis to accelerate PF. ROS can induce the activation of AKT/GSK3ß signaling, and inhibition of it can inhibit ROS-induced inflammation and EMT by the NF-kB/NLRP3 axis, thereby inhibiting PF. CD-NPs induced PF by promoting inflammation and EMT via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3ß signals. This study provides a valuable experimental basis for the prevention and treatment of coal workers' pneumoconiosis. Illustration of the overall research idea of this study: IGF1 stimulates coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and EMT via the NF-κB/NLRP3 pathway driven by ROS-mediated AKT/GSK3ß signals.

Differences in the characteristics and pulmonary toxicity of nano- and micron-sized respirable coal dust.

BACKGROUND: The characteristics of coal dust (CD) particles affect the inhalation of CD, which causes coal worker's pneumoconiosis (CWP). CD nanoparticles (CD-NPs, < 500 nm) and micron particles (CD-MPs, < 5 µm) are components of the respirable CD. However, the differences in physicochemical properties and pulmonary toxicity between CD-NPs and CD-MPs remain unclear. METHODS: CD was analyzed by scanning electron microscopy, Malvern nanoparticle size potentiometer, energy dispersive spectroscopy, infrared spectroscopy, and electron paramagnetic resonance spectroscopy. CCK-8 assay, ELISA, transmission electron microscope, JC-1 staining, reactive oxygen species activity probe, calcium ion fluorescent probe, AO/EB staining, flow cytometry, and western blot were used to determine the differences between CD-NPs and CD-MPs on acute pulmonary toxicity. CCK-8, scratch healing and Transwell assay, hematoxylin-eosin and Masson staining, immunohistochemistry, immunofluorescence, and western blot were applied to examine the effects of CD-NPs and CD-MPs on pneumoconiosis. RESULTS: Analysis of the size distribution of CD revealed that the samples had been size segregated. The carbon content of CD-NPs was greater than that of CD-MPs, and the oxygen, aluminum, and silicon contents were less. In in vitro experiments with A549 and BEAS-2B cells, CD-NPs, compared with CD-MPs, had more inflammatory vacuoles, release of pro-inflammatory cytokines (IL-6, IL-1ß, TNFα) and profibrotic cytokines (CXCL2, TGFß1), mitochondrial damage (reactive oxygen species and Ca2+ levels and decreased mitochondrial membrane potential), and cell death (apoptosis, pyroptosis, and necrosis). CD-NPs-induced fibrosis model cells had stronger proliferation, migration, and invasion than did CD-MPs. In in vivo experiments, lung coefficient, alveolar inflammation score, and lung tissue fibrosis score (mean: 1.1%, 1.33, 1.33) of CD-NPs were higher than those of CD-MPs (mean: 1.3%, 2.67, 2.67). CD-NPs accelerated the progression of pulmonary fibrosis by upregulating the expression of pro-fibrotic proteins and promoting epithelial-mesenchymal transition. The regulatory molecules involved were E-cadherin, N-cadherin, COL-1, COL-3, ZO-1, ZEB1, Slug, α-SMA, TGFß1, and Vimentin. CONCLUSIONS: Stimulation with CD-NPs resulted in more pronounced acute and chronic lung toxicity than did stimulation with CD-MPs. These effects included acute inflammatory response, mitochondrial damage, pyroptosis, and necrosis, and more pulmonary fibrosis induced by epithelial-mesenchymal transition.

ASIC1α up-regulates MMP-2/9 expression to enhance mobility and proliferation of liver cancer cells via the PI3K/AKT/mTOR pathway.

A major challenge in the treatment of liver cancer is that a large proportion of patients fail to achieve long-term disease control, with death from liver cancer cell migration and invasion. Acid-sensitive ion channel 1α (ASIC1α) is involved in the migration, invasion, and proliferation of liver cancer cells. Therefore, we explored the mechanism of ASIC1α-mediated liver cancer cell migration and invasion. We determined the levels of ASIC1α by western blotting and immunofluorescence in HepG2 and SK-Hep1 cells cultured in various acidic conditions. In addition, wound healing assay, transwell invasion assay, and MTT assay were conducted to assess the migration, invasion, and proliferation abilities of liver cancer cells. Western blotting was conducted to determine the levels of MMP2, MMP9, ASIC1α, p-PI3Kp85, t-PI3Kp85, p-AKT(Ser473), t-AKT, p-mTOR (Ser2448), t-mTOR. We first found that the levels of ASIC1α in the HepG2 and SK-Hep1 cells in acidic conditions (pH 6.5) were significantly increased. Inhibition and knockdown of ASIC1α down-regulated MMP-2/9 expression and inhibited the migration, invasion, and proliferation of HepG2 and SK-Hep1 cells; overexpression of ASIC1α had the opposite effect. We further demonstrated that ASIC1α up-regulates MMP-2/9 via activation of the PI3K/AKT/mTOR pathway, thereby promoting migration, invasion, and proliferation of liver cancer cells. Overexpression of MMP-2/9 and activation of AKT reversed these effects on liver cancer cells caused by inhibition of ASIC1α. We conclude that ASIC1α can regulate migration, invasion, and proliferation of liver cancer cells through the MMP-2/9/PI3K/AKT/mTOR pathway. These observations may provide a new reference for liver cancer chemotherapy.

The effects of coal dust exposure on DNA damage and repair of human bronchial epithelial cells.

To explore the effects of coal dust exposure on DNA damage and repair, human bronchial epithelial BEAS-2B cells were exposed to coal dust and the cellular response was investigated. It was found that γ-H2AX foci of DNA damage appeared, γ-H2AX protein level increased, and the rate of cell apoptosis was significantly elevated when BEAS-2B cells were exposed to coal dust for a short time. Phagocytized coal dust particles, swollen mitochondria, and reduced mitochondrial membrane potential were simultaneously identified. Moreover, Caspase-9, Caspase-3, and DFF45 proteins of the mitochondrial apoptotic pathway were activated. After the cells were exposed to coal dust chronically, phosphorylation levels of DNA repair kinases (ATM/ATR, DNA-PKcs) and downstream regulatory protein AKT were significantly upregulated. γ-H2AX foci and tail DNA of the cells following treatment with cisplatin were also reduced, and the colony formation rate was improved. It was concluded that coal dust could induce DNA damage, cause mitochondrial depolarization, and activate mitochondrial apoptosis pathways in BEAS-2B cells. Additionally, activated DNA repair kinases (ATM/ATR and DNA-PKcs) and their regulatory protein AKT increased DNA repair and proliferation of BEAS-2B cells chronically exposed to coal dust.

ASIC1a stimulates the resistance of human hepatocellular carcinoma by promoting EMT via the AKT/GSK3ß/Snail pathway driven by TGFß/Smad signals.

Multidrug resistance is the main obstacle to curing hepatocellular carcinoma (HCC). Acid-sensing ion channel 1a (ASIC1a) has critical roles in all stages of cancer progression, especially invasion and metastasis, and in resistance to therapy. Epithelial to mesenchymal transition (EMT) transforms epithelial cells into mesenchymal cells after being stimulated by extracellular factors and is closely related to tumour infiltration and resistance. We used Western blotting, immunofluorescence, qRT-PCR, immunohistochemical staining, MTT, colony formation and scratch healing assay to determine ASIC1a levels and its relationship to cell proliferation, migration and invasion. ASIC1a is overexpressed in HCC tissues, and the amount increased in resistant HCC cells. EMT occurred more frequently in drug-resistant cells than in parental cells. Inactivation of ASIC1a inhibited cell migration and invasion and increased the chemosensitivity of cells through EMT. Overexpression of ASIC1a upregulated EMT and increased the cells' proliferation, migration and invasion and induced drug resistance; knocking down ASIC1a with shRNA had the opposite effects. ASIC1a increased cell migration and invasion through EMT by regulating α and ß-catenin, vimentin and fibronectin expression via the AKT/GSK-3ß/Snail pathway driven by TGFß/Smad signals. ASIC1a mediates drug resistance of HCC through EMT via the AKT/GSK-3ß/Snail pathway.

A pan-cancer analysis on the carcinogenic effect of human adenomatous polyposis coli.

Adenomatous polyposis coli (APC) is the most commonly mutated gene in colon cancer and can cause familial adenomatous polyposis (FAP). Hypermethylation of the APC promoter can also promote the development of breast cancer, indicating that APC is not limited to association with colorectal neoplasms. However, no pan-cancer analysis has been conducted. We studied the location and structure of APC and the expression and potential role of APC in a variety of tumors by using The Cancer Genome Atlas and Gene Expression Omnibus databases and online bioinformatics analysis tools. The APC is located at 5q22.2, and its protein structure is conserved among H. sapiens, M. musculus with C. elaphus hippelaphus. The APC identity similarity between homo sapiens and mus musculus reaches 90.1%. Moreover, APC is highly specifically expressed in brain tissues and bipolar cells but has low expression in most cancers. APC is mainly expressed on the cell membrane and is not detected in plasma by mass spectrometry. APC is low expressed in most tumor tissues, and there is a significant correlation between the expressed level of APC and the main pathological stages as well as the survival and prognosis of tumor patients. In most tumors, APC gene has mutation and methylation and an enhanced phosphorylation level of some phosphorylation sites, such as T1438 and S2260. The expressed level of APC is also involved in the level of CD8+ T-cell infiltration, Tregs infiltration, and cancer-associated fibroblast infiltration. We conducted a gene correlation study, but the findings seemed to contradict the previous analysis results of the low expression of the APC gene in most cancers. Our research provides a comparative wholesale understanding of the carcinogenic effects of APC in various cancers, which will help anti-cancer research.

Autophagy up-regulated by MEK/ERK promotes the repair of DNA damage caused by aflatoxin B1.

Aflatoxin B1 (AFB1), a kind of mycotoxin, exerts its cytotoxicity by increasing the oxidative damage of target organs, especially the liver. In vivo and in vitro experiments were carried out to elucidate the toxic mechanism of AFB1. The results of MTT, cloning-formation, flow cytometry, immunocytochemistry, Reverse transcription PCR (RT-PCR) and western blot showed that AFB1 activated NOX2 gp91 phox, inhibited proliferation and migration, and blocked cell cycle at G0/G1 period of HHL-5 cells. Autophagy promoted the repair of NOX2-dependent DNA damage. NOX2/gp91 phox mainly activates MEK/ERK pathway and then up-regulates autophagy. In vivo experiments have shown that AFB1 (0.75 mg/kg daily orally, 4 weeks) had no significant changes in the size and shape of the liver in mice. However, these treatments lead to structural abnormalities of hepatocytes and DNA damage. In summary, AFB1 caused intracellular oxidative stress and DNA damage, NOX2/gp91-phox activates the MEK/ERK pathway, and upregulated autophagy to promote the repair of DNA damage. We concluded that by increasing the level of autophagy, the ability of anti-AFB1 toxicity of liver can be increased.

PKI-587 enhances radiosensitization of hepatocellular carcinoma by inhibiting the PI3K/AKT/mTOR pathways and DNA damage repair.

Radiation is an important therapeutic strategy for hepatocellular (HCC). In this study, we evaluated the role of the dual PI3K/mTOR inhibitor, PKI-587, on radiosensitization of HCC and its possible mechanism. MTT, colony formation, flow cytometry, and immunofluorescence were used to analyze the proliferation, cell cycle, formation of residual γ-H2AX foci, and apoptosis of HCC cells. A SK-Hep1 xenograft HCC model was used to assess the effects of PKI-587 in combination with ionizing radiation in vivo. The activation levels of PI3K/AKT/mTOR and DNA damage repair pathways and their downstream effector molecules were detected with Western blot. It was found that PKI-587 sensitized HCC cells to radiation by increasing DNA damage, enhancing G0/G1 cell-cycle arrest, and inducing apoptosis. In vivo, the combination of radiation with PKI-587 significantly inhibited tumor growth. These findings suggest the usefulness of PKI-587 on radiosensitization of HCC cells by inhibiting the PI3K/AKT/mTOR and DNA damage repair pathways. The combination of ionizing radiation and PKI-587 may be a strategy to improve the efficacy of treating HCC.

Curcumin reverses hepatic epithelial mesenchymal transition induced by trichloroethylene by inhibiting IL-6R/STAT3.

OBJECTIVE: Epithelial mesenchymal transition (EMT) and inflammation have been identified as carcinogenic agents. This study aims to investigate whether inhibition of trichloroethylene (TCE) associated hepatocellular carcinoma (HCC) by curcumin is associated with inflammation and EMT. METHODS: In the current study, TCE sub-chronic cell model was induced in vitro, and the effects of TCE on cell proliferation, migration, invasion, and expression of functional proteins were verified by Western blot, MTT, clone formation, wound healing, Transwell. The detoxification effect of curcumin on TCE was explored by a mouse tumor-bearing experiment. RESULTS: TCE induces hepatocyte migration, colony formation, and EMT in vitro. In vivo studies have shown that curcumin significantly reduces the mortality of mice and control the occurrence and size of liver tumors by inhibiting the IL-6/STAT3 signaling pathway. In vitro, curcumin inhibits the proliferation of HepG2 cells as determined by MTT assay. In addition, curcumin significantly inhibited the protein expression of IL-6R, STAT3, snail, survivin, and cyclin D1 in THLE-2 and HepG2 cells induced by IL-6. CONCLUSION: Curcumin has anti-inflammatory and anti-proliferative effects, and inhibits the development of HCC induced by TCE by reversing IL-6/STAT3 mediated EMT.

LY­294002 enhances the chemosensitivity of liver cancer to oxaliplatin by blocking the PI3K/AKT/HIF­1α pathway.

Liver cancer remains one of the leading causes of cancer deaths worldwide. The therapeutic effect of oxaliplatin on liver cancer is often limited by acquired resistance of the cancer cells. Abnormal activation of the PI3K/AKT pathway plays an important role in the acquired resistance of oxaliplatin. The present study investigated the effects of the PI3K inhibitor LY­294002 and AKT inhibitor MK2206 on the chemosensitivity of oxaliplatin­resistant liver cancer cells and the molecular mechanism involved. An oxaliplatin­resistant liver cancer cell line HepG2R was developed. MTT assay, clone formation experiments, flow cytometry and Annexin V­FITC/PI staining were used to determine the proliferation, cycle and apoptosis of HepG2R cells when oxaliplatin was combined with LY­294002 or MK2206 treatment. The effects of LY­294002 and MK­2206 on the abnormal activation of PI3K/AKT pathway and hypoxia inducible factor (HIF)­1α protein level in HepG2R cells were detected using western blotting. The results indicated that the PI3K/AKT pathway is stably activated in HepG2R cells. Compared with the AKT inhibitor MK2206, the PI3K inhibitor LY­294002 more effectively downregulated the phosphorylation levels of p85, p110α, p110ß, p110γ and AKT in the PI3K/AKT pathway in HepG2R cells, and more effectively inhibited the proliferation of the cells. LY­294002 enhanced the chemotherapy sensitivity of HepG2R cells to oxaliplatin by inducing G0/G1 phase arrest and increasing the proportion of apoptotic cells. In addition, LY­294002 reduced the level of HIF­1α, which is highly expressed in HepG2R cells. It was concluded that LY­294002 enhanced the chemosensitivity of liver cancer cells to oxaliplatin by inhibiting the PI3K/AKT signaling pathway, which may be related to the inhibition of HIF­1α expression. These findings may have clinical significance for the treatment of oxaliplatin­resistant liver cancer.

BEZ235 Increases the Sensitivity of Hepatocellular Carcinoma to Sorafenib by Inhibiting PI3K/AKT/mTOR and Inducing Autophagy.

Acquired resistance of hepatocellular carcinoma (HCC) to sorafenib (SFB) is the main reason for the failure of SFB treatment of the cancer. Abnormal activation of the PI3K/AKT/mTOR pathway is important in the acquired resistance of SFB. Therefore, we investigated whether BEZ235 (BEZ) could reverse acquired sorafenib resistance by targeting the PI3K/mTOR pathway. A sorafenib-resistant HCC cell line Huh7R was established. MTT assay, clone formation assay, flow cytometry, and immunofluorescence were used to analyze the effects of BEZ235 alone or combined with sorafenib on cell proliferation, cell cycle, apoptosis, and autophagy of Huh7 and Huh7R cells. The antitumor effect was evaluated in animal models of Huh7R xenografts in vivo. Western blot was used to detect protein levels of the PI3K/AKT/mTOR pathway and related effector molecules. In vitro results showed that the Huh7R had a stronger proliferation ability and antiapoptosis effect than did Huh7, and sorafenib had no inhibitory effect on Huh7R. SFB + BEZ inhibited the activation of the PI3K/AKT/mTOR pathway caused by sorafenib. Moreover, SFB + BEZ inhibited the proliferation and cloning ability, blocked the cell cycle in the G0/G1 phase, and promoted apoptosis in the two cell lines. The autophagy level in Huh7R cells was higher than in Huh7 cells, and BEZ or SFB + BEZ further promoted autophagy in the two cell lines. In vivo, SFB + BEZ inhibited tumor growth by inducing apoptosis and autophagy. We concluded that BEZ235 enhanced the sensitivity of sorafenib through suppressing the PI3K/AKT/mTOR pathway and inducing autophagy. These observations may provide the experimental basis for sorafenib combined with BEZ235 in trial treatment of HCC.

Oxaliplatin and Gedatolisib (PKI-587) Co-Loaded Hollow Polydopamine Nano-Shells with Simultaneous Upstream and Downstream Action to Re-Sensitize Drugs-Resistant Hepatocellular Carcinoma to Chemotherapy.

Multidrug resistance (MDR) is a key to the ineffectiveness of hepatocellular carcinoma (HCC) chemotherapy. Oxaliplatin (OXA), as one of the first-line chemotherapeutic drugs for HCC, abnormally activates the PI3K/AKT/mTOR signaling pathway and DNA damage repair pathway (NHEJ and HR), causing drug resistance and consequnet compromised efficacy. Herein, we developed a hollow polydopamine nanoparticle (H-PDA)-based nano-delivery system (O/P-HP) that contained OXA and a dual PI3K/mTOR inhibitor PKI-587 with complementary effects for combating drug resistance in cancer chemotherapy. The hollow structure of H-PDA endowed O/P-HP with high loading efficiencies of OXA and PKI-587-up to 49.6% and 7.0%, respectively. In addition, benefiting from the intracellular delivery of H-PDA as well as the highly concentrated drugs therein, O/P-HP inhibited the proliferation of OXA-resistant HR cells, resulting in a cell viability of only 17.63%. These values were significantly superior to those with OXA single-agent treatment and treatment with free OXA in combination with PKI-587. We examined the intrinsic mechanisms of the combination therapy: O/PHP had excellent anti-cancer effects via the simultaneous upstream and downstream action to re-sensitize HR cells to chemotherapy; OXA induced strong apoptosis via the direct platinum lesions on DNA molecules, while PKI-587 normalized the abnormally activated PI3K/AKT/mTOR signaling pathway and DNA damage repair pathway (NHEJ and HR) that could attenuate the effectiveness of OXA, thus resulting in inhibition of cell proliferation, migration and DNA repair enzyme activity and the augment of apoptotic effects. Such combination therapy, with simultaneous upstream and downstream action, may be a strategy for minimizing resistance for anti-cancer treatments.

LY3214996 relieves acquired resistance to sorafenib in hepatocellular carcinoma cells.

Background: Sorafenib, an oral multi-kinase inhibitor of rapidly accelerated fibrosarcoma; vascular endothelial growth factor receptor-2/3, platelet-derived growth factor receptor, c-Kit, and Flt-3 signaling, is approved for treatment of advanced hepatocellular carcinoma (HCC). However, the benefit of sorafenib is often diminished because of acquired resistance through the reactivation of ERK signaling in sorafenib-resistant HCC cells. In this work, we investigated whether adding LY3214996, a selective ERK1/2 inhibitor, to sorafenib would increase the anti-tumor effectiveness of sorafenib to HCC cells. Methods: The Huh7 cell line was used as a cell model for treatment with sorafenib, LY3214996, and their combination. Phosphorylation of the key kinases in the Ras/Raf/MAPK and PI3K/Akt pathways, protein expression of the cell cycle, and apoptosis migration were assessed with western blot. MTT and colony-formation assays were used to evaluate cell proliferation. Wound-healing assay was used to assess cell migration. Cell cycle and apoptosis analyses were conducted with flow cytometry. Results: LY3214996 decreased phosphorylation of the Ras/Raf/MAPK and PI3K/Akt pathways, including p-c-Raf, p-P90RSK, p-S6K and p-eIF4EBP1 activated by sorafenib, despite increased p-ERK1/2 levels. LY3214996 increased the anti-proliferation, anti-migration, cell-cycle progression, and pro-apoptotic effects of sorafenib on Huh7R cells. Conclusions: Reactivation of ERK1/2 appears to be a molecular mechanism of acquired resistance of HCC to sorafenib. LY3214996 combined with sorafenib enhanced the anti-tumor effects of sorafenib in HCC. These findings form a theoretical basis for trial of LY3214996 combined with sorafenib as second-line treatment of sorafenib-resistant in advanced HCC.

hGC33-Modified and Sorafenib-Loaded Nanoparticles have a Synergistic Anti-Hepatoma Effect by Inhibiting Wnt Signaling Pathway.

Delivery of tumor-specific inhibitors is a challenge in cancer treatment. Antibody-modified nanoparticles can deliver their loaded drugs to tumor cells that overexpress specific tumor-associated antigens. Here, we constructed sorafenib-loaded polyethylene glycol-b-PLGA polymer nanoparticles modified with antibody hGC33 to glypican-3 (GPC3 +), a membrane protein overexpressed in hepatocellular carcinoma. We found that hGC33-modified NPs (hGC33-SFB-NP) targeted GPC3+ hepatocellular carcinoma (HCC) cells by specifically binding to GPC3 on the surface of HCC cells, inhibited Wnt-induced signal transduction, and inhibited HCC cells in G0/1 by down-regulating cyclin D1 expression, thus attenuating HCC cell migration by inhibiting epithelial-mesenchymal transition. hGC33-SFB-NP inhibited the migration, cycle progression, and proliferation of HCC cells by inhibiting the Ras/Raf/MAPK pathway and the Wnt pathway in tandem with GPC3 molecules, respectively. hGC33-SFB-NP inhibited the growth of liver cancer in vivo and improved the survival rate of tumor-bearing mice. We conclude that hGC33 increases the targeting of SFB-NP to HCC cells. hGC33-SFB-NP synergistically inhibits the progression of HCC by blocking the Wnt pathway and the Ras/Raf/MAPK pathway.

Gefitinib sensitization of cisplatin-resistant wild-type EGFR non-small cell lung cancer cells.

PURPOSE: The usual first-line strategy of wild-type EGFR (wtEGFR) non-small cell lung cancer (NSCLC) remains cisplatin-based chemotherapy. However, cisplatin often loses effectiveness because most tumors acquire drug resistance over time. As EGFR is the most important pro-survival/proliferation signal receptor in NSCLC cells, we aimed at investigating whether cisplatin resistance is related to EGFR activation and further evaluating the combined effects of cisplatin/gefitinib (EGFR-tyrosine kinase inhibitor, EGFR-TKI) on cisplatin-resistant wtEGFR NSCLC cells. MATERIALS AND METHODS: EGFR activation was analysed in parental and cisplatin-resistant wtEGFR NSCLC cell lines (H358 and H358R, A549 and A549R). Cellular proliferation and apoptosis of H358R/A549R cells treated with cisplatin or gefitinib, alone or in combination were investigated, and the related effector protein was detected by western blot analysis. Anti-tumor effect of two drugs combined was evaluated in animal models of H358R xenografts in vivo. RESULTS: EGFR was significantly phosphorylated in cisplatin-resistant wtEGFR NSCLC cells H358R and A549R than their parental cells. In H358R and A549R cells, anti-proliferative ability of gefitinib was further improved, and gefitinib combined with cisplatin enhanced inhibition of cellular survive/proliferation, and promotion of apoptosis in vitro. The combined effects were also associated with the inhibition of EGFR downstream effector proteins. Similarly, in vivo, gefitinib and cisplatin in combination significantly inhibited tumor growth of H358R xenografts. CONCLUSION: Abnormal activation of EGFR may induce wtEGFR NSCLC cell resistance to cisplatin. The combined effects of cisplatin/gefitinib suggest that gefitinib, as a combination therapy for patients with cisplatin-resistant wtEGFR NSCLC should be considered.

The PI3K/mTOR dual inhibitor BEZ235 nanoparticles improve radiosensitization of hepatoma cells through apoptosis and regulation DNA repair pathway.

Polymer materials encapsulating drugs have broad prospects for drug delivery. We evaluated the effectiveness of polyethylene glycol-poly (lactic-co-glycolic acid) (PLGA-PEG) encapsulation and release characteristics of PI3K/mTOR inhibitor NVP-BEZ235 (BEZ235). We proposed a strategy for targeting radiosensitization of liver cancer cells. The biocompatibility, cell interaction, and internalization of Glypican-3 (GPC3) antibody-modified, BEZ235-loaded PLGA-PEG nanoparticles (NP-BEZ235-Ab) in hepatoma cells in vitro were studied. Also, the cell killing effect of NP-BEZ235-Ab combined with γ-ray cell was evaluated. We used confocal microscopy to monitor nanoparticle-cell interactions and cellular uptake, conducted focus-formation experiments to analyze the synergistic biological effects of NP-BEZ235-Ab and priming, and studied synergy in liver cancer cells using molecular biological methods such as western blotting. We found that PLGA-PEG has good loading efficiency for BEZ235 and high selectivity to GPC3-positive HepG2 liver cancer cells, thus documenting that NP-BEZ235-Ab acts as a small-molecule drug delivery nanocarrier. At the nominal concentration, the NP-BEZ235-Ab nanoformulation synergistically kills liver cancer cells with significantly higher efficiency than does the free drug. Thus, NP-BEZ235-Ab is a potential radiosensitizer.

Resistance of hepatocellular carcinoma to sorafenib can be overcome with co-delivery of PI3K/mTOR inhibitor BEZ235 and sorafenib in nanoparticles.

Background: The combination of BEZ235 with sorafenib (SFB) enhances anti-hepatocellular carcinoma (HCC) efficacy of the two agents. However, pharmacokinetic profiles in vivo and different endocytosis abilities of these two drugs hinder their therapeutic application.Research design and methods: In this work, we developed d-α-tocopheryl polyethylene glycol 1000 succinate - polycaprolactone polymer nanoparticles (NPs) for co-delivery of SFB and BEZ235 (SFB/BEZ235-NPs). Explored the anti-proliferative and pro-apoptotic effects of SFB/BEZ235-NPs through in vitro and in vivo experiments.Results: Stabilized SFB/BEZ235-NPs were prepared with optimized drug ratio, yielding high encapsulation efficiency, low polydispersity, and enhanced cellular internalization in HepG2 cells. Synergistic cytotoxicity and pro-apoptotic ability were documented. In vivo pharmacokinetic results revealed extended circulation and bioavailability of SFB/BEZ235-NPs compared with those of free drugs. SFB/BEZ235-NPs enhanced antitumor effectiveness in SFB-resistant HCC xenograft mouse models.Conclusion: Taken together, the results of this study describe a promising strategy using SFB and BEZ235 in a nanoparticle formulation for treatment of SFB-resistant HCC.