Discovery of Novel Mono-Carbonyl Curcumin Derivatives as Potential Anti-Hepatoma Agents.

Curcumin possesses a wide spectrum of liver cancer inhibition effects, yet it has chemical instability and poor metabolic properties as a drug candidate. To alleviate these problems, a series of new mono-carbonyl curcumin derivatives G1-G7 were designed, synthesized, and evaluated by in vitro and in vivo studies. Compound G2 was found to be the most potent derivative (IC50 = 15.39 µM) compared to curcumin (IC50 = 40.56 µM) by anti-proliferation assay. Subsequently, molecular docking, wound healing, transwell, JC-1 staining, and Western blotting experiments were performed, and it was found that compound G2 could suppress cell migration and induce cell apoptosis by inhibiting the phosphorylation of AKT and affecting the expression of apoptosis-related proteins. Moreover, the HepG2 cell xenograft model and H&E staining results confirmed that compound G2 was more effective than curcumin in inhibiting tumor growth. Hence, G2 is a promising leading compound with the potential to be developed as a chemotherapy agent for hepatocellular carcinoma.

Dynamics simulation and in vitro studies of betulinic acid derivative with liver X receptor.

Molecular dynamics simulation of the dominant conformational conjugate was performed for 40 ns and 100 ns via Amber software based on molecular docking by Sybyl software. Because the RMSD and RMSF of 100 ns MD simulation were higher than that of 40 ns MD simulation, the 40 ns was reasonable and credible for MD simulation. The binding free energy and decomposition free energy of the two systems of betulinic acid, com3 with liver X receptor was calculated by the MM_GBSA and MM_PBSA methods, respectively. The results showed that the two systems reached equilibrium and convergence at 20 ns, both stable at about 2 Å, and exhibited low volatility in the range of amino acid 270 to 370 (RMSF <1 Å). The binding energy of com3 (ΔGbind = -68.02 kcal/mol by the MM_GBSA method or -55.50 kcal/mol by the MM_PBSA method) with the liver X receptor was lower than that of betulinic acid (ΔGbind = -55.70 kcal/mol or -42.73 kcal/mol) respectively, and van der Waals force was the most important main driving force, which was consistent with molecular docking and previous experiments. Hydrophobic groups and aromatic rings can be introduced appropriately in structure optimization to increase the van der Waals force and π-π accumulation effect of betulinic acid and liver X receptor, which is conducive to binding and thereby increasing antitumor activity. The clone formation assay and results of western blotting indicated that BA derivative com3 exposure inhibited cell proliferation may relate to the regulation of the AKT/mTOR pathway in 7721 cells. This study clarifies the dynamic interaction mode and potential mechanism of betulinic acid and its derivatives with the liver X receptor, which provides a new idea for the rapid screening of liver X receptor agonists from traditional Chinese medicines.Communicated by Ramaswamy H. Sarma.

Design, Synthesis, and Antitumor Evaluation of Novel Mono-Carbonyl Curcumin Analogs in Hepatocellular Carcinoma Cell.

Curcumin is a polyphenolic natural product that has promising anticancer properties. However, its clinical utility is limited by its chemical instability and poor metabolic properties. In this paper, a series of new curcumin analogs were synthesized and found to be potent antiproliferative agents against the HepG2 cell line by MTT assay. In general, Group B with single ketone and group C with chalcone were markedly more cytotoxic than group A with diketone. Compound B5 was found as the most potent analog (IC50 = 11.33 µM) compared to curcumin (IC50 = 32.83 µM) and the mechanism of its cytotoxicity was investigated. The result of the wound healing assay indicated B5 strong potential to suppress HepG2 cell migration in a dose- and time-dependent manner. Subsequent assays (including JC-1 staining, Bcl-2, and caspase 3 protein levels by Western blotting) confirmed that B5 exposure induced apoptosis in HepG2 cells. Curcumin-induced comprehensive transcriptomes profile, Western blotting, molecular docking, and molecular dynamics analysis showed that the mechanism may relate to the regulation of cellular metabolic process and the expression of AKT protein. Taken together, we could conclude that curcumin and its analogs induced HepG2 cell proliferation, migration, and apoptosis via AKT signaling pathway and the mitochondrial death pathway. This study could lay the foundation for optimizing curcumin and provide valuable information for finding novel anti-HCC drugs.

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.

Aflatoxin B1: metabolism, toxicology, and its involvement in oxidative stress and cancer development.

Aflatoxins are a class of carcinogenic mycotoxins produced by Aspergillus fungi, which are widely distributed in nature. Aflatoxin B1 (AFB1) is the most toxic of these compounds and its metabolites have a variety of biological activities, including acute toxicity, teratogenicity, mutagenicity and carcinogenicity, which has been well-characterized to lead to the development of hepatocellular carcinoma (HCC) in humans and animals. This review focuses on the metabolism of AFB1, including epoxidation and DNA adduction, as it concerns the initiation of cancer and the underlying mechanisms. In addition to DNA adduction, inflammation and oxidative stress caused by AFB1 can also participate in the occurrence of cancer. Therefore, the main carcinogenic mechanism of AFB1 related ROS is summarized. This review also describes recent reports of AFB1 exposures in occupational settings. It is hoped that people will pay more attention to occupational health, in order to reduce the incidence of cancer caused by occupational exposure.

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.

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.

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.

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.

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.

An overview of autophagy: Mechanism, regulation and research progress.

Autophagy refers to the formation of autophagosomes by membrane wrapping part of the cytoplasm and the organelles and proteins that need to be degraded in the cells. Autophagosomes are fused with lysosomes to form autophagolysosome, which degrade the contents of the inclusions, to achieve cell homeostasis and organelle renewal. The regulatory mechanism of autophagy is complex, and its upstream signaling pathway mainly involves mTOR dependent pathway and mTOR independent pathway (AMPK, PI3K, Ras-MAPK, p53, PTEN, endoplasmic reticulum stress). Autophagy is a phenomenon of "self-eating" in cells. Apoptosis is a phenomenon of "self-killing". Both of them share the same stimulating factors and regulatory proteins, but the threshold of induction is different. How to transform and coordinate is not clear at present. This paper summarizes the history of autophagy discovery, the structure and function of related molecules, the biological function of autophagy, the regulatory mechanism and the research results of the relationship between autophagy and apoptosis.

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.

Anti-GPC3 Antibody-Conjugated BEZ235 Loaded Polymeric Nanoparticles (Ab-BEZ235-NP) Enhances Radiosensitivity in Hepatocellular Carcinoma Cells by Inhibition of DNA Double-Strand Break Repair.

AIM: To assess AB-BEZ235-NP potential as a radio-sensitizer in hepatocellular carcinoma models. METHOD: By comparing hepatocellular carcinoma cell with simple radiation or combined AB-BEZ235-NP therapy, the HCC apoptosis and self-repair level have significant differences in mortality rates and cell migration abilities. RESULTS: Cell proliferation and DNA damage increased by pretreatment with AB-BEZ235-NP after irradiation; further studies on the repair pathway indicated that AB-BEZ235-NP inhibited the important pathway of DSB repair. Our results further show that AB-BEZ235-NP significantly inhibits the phosphorylation of the canonical protein, γ-H2AX, in the NHEJ DSB repair pathway and Serine Protein Kinase (SPK) ATM, and TP53-Binding Protein one. More importantly, AB-BEZ235-NP increased the mount of mean γ-H2AX Foci in irradiated cells, indicating that AB-BEZ235-NP can selectively inhibit DSB repair in HCC cells. Therefore, these results clearly eludicate that treatment with AB-BEZ235-NP is a potential promising therapy which can increase the radiosensitivity to HCC.

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.

Increased efficacy of gefitinib on cisplatin-resistant wild-type epidermal growth factor receptor non-small cell lung cancer cells.

BACKGROUND: Gefitinib is a first-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that has become first-line treatment for patients with mutant EGFR non-small cell lung cancer (NSCLC). Despite its anti-tumor activity, the benefit of gefitinib in patients with wild-type EGFR NSCLC is debated. This work aimed to evaluate the effects of gefitinib on cisplatin-resistant wild-type EGFR NSCLC cells in in vitro and in vivo animal xenografts. METHODS: We established a cisplatin-resistant wild-type EGFR NSCLC cell line, H358R, to evaluate the cells' sensitivity to gefitinib compared with that of parental cell line H358. We first tested the p-EGFR of gefitinib's target in H358R and H358 cell line by western blot and immunofluorescence. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clone formation assay, flow cytometry and annexin V-fluorescein/propidium iodide staining were used to investigate cellular proliferation and apoptosis of H358R/H358 cells treated with gefitinib, and the anti-tumor effect was evaluated in female BALB/c nude mice models of xenografts in vivo. RESULTS: EGFR and the downstream node molecules ERK and AKT were significantly more phosphorylated in H358R than in the parental cells and were inhibited by gefitinib. In H358R cells, gefitinib increased the inhibition of cell survival/proliferation, and the promotion of apoptosis in vitro. The increased anti-tumor effect was present also in H358R xenografts in vivo. CONCLUSIONS: Abnormal activation of EGFR in H358R cells results in enhanced sensitivity to gefitinib. The improved efficacy of gefitinib on cisplatin-resistant wild-type EGFR NSCLC cells suggests that gefitinib as sequential therapy for patients with cisplatin-resistant wild-type EGFR NSCLC should be considered.

BEZ235 increases sorafenib inhibition of hepatocellular carcinoma cells by suppressing the PI3K/AKT/mTOR pathway.

BACKGROUND: Sorafenib is an oral multi-kinase inhibitor that inhibits hepatocellular carcinoma (HCC) via the Ras/Raf/MAPK pathway. However, sorafenib loses effectiveness because most tumors acquire drug resistance over time. As the PI3K/AKT/mTOR signaling pathway is also activated abnormally in HCC, we evaluated the effect of sorafenib, in combination with a dual PI3K/mTOR inhibitor, BEZ235, on HCC cell proliferation and survival in vitro. MATERIALS AND METHODS: Biological phenotypes were analysed in HCC cell lines, parental and sorafenib-resistant HepG2 cells (HepG2 and HepG2R), treated with sorafenib or BEZ235, alone or in combination. HCC cellular proliferation and apoptosis were investigated, and perturbations of the Ras/Raf/MAPK and PI3K/AKT/mTOR signaling/survival pathways were evaluated by western blot analysis. RESULTS: BEZ235 enhanced sorafenib inhibition of cellular proliferation, migration, and promotion of apoptosis in HepG2 and HepG2R cells. The combined effects were associated with inhibition of phosphorylation of AKT, mTOR and S6K in the PI3K/AKT/mTOR pathway, whereas the combination of sorafenib and BEZ235 did not significantly alter the Ras/Raf/MAPK pathway compared with the effect of sorafenib alone. CONCLUSION: Sorafenib/BEZ235 combination has potent anti-HCC cell activity. This anti-tumor activity is most likely multi-factorial, mainly involving PI3K down-regulation and AKT, mTOR and S6K dephosphorylation. Combined inhibition of PI3K/AKT/mTOR and Ras/Raf/MAPK pathways enhances sorafenib inhibition of HCC. The results of these in vitro studies suggest that trials of combined sorafenib and BEZ235 in the treatment of HCC should be considered.

PKI-587 enhances chemosensitivity of oxaliplatin in hepatocellular carcinoma through suppressing DNA damage repair pathway (NHEJ and HR) and PI3K/AKT/mTOR pathway.

Oxaliplatin resistance limits its effectiveness in the treatment of hepatocellular carcinoma (HCC). Abnormal activation of the PI3K/AKT/mTOR pathway has been associated with decreased survival of HCC patients, anti-apoptosis after chemotherapeutic drug-induced DNA damage, and chemoresistance. In this research, we evaluated the effect of the dual PI3K/mTOR inhibitor, PKI-587, on the sensitivity of oxaliplatin in HCC. Two HCC cell lines (HepG2 and SK-Hep1) were used to analyze PKI-587 for DNA damage response, cell proliferation, clonogenic survival, cell cycle and apoptosis after oxaliplatin treatment. A HepG2 tumor-bearing model was used to assess the in vivo effects of the combination of the two compounds. In HCC cells, oxaliplatin stably activated the PI3K/AKT/mTOR pathway, including up-regulation of p-Akt (Ser473), p-mTOR (Ser2448), p-mTOR (Ser2481), p-elF4EBP1, and p-S6K1, and activated the DNA damage repair pathways (non-homologous end joining (NHEJ) and homologous recombination (HR)), up-regulation of p-DNAPKcs (Ser2056), p-ATM (Ser1981), and p-ATR (Ser428), which were attenuated by PKI-587. Compared with oxaliplatin alone, the combination of PKI-587 and oxaliplatin increased the number of γ-H2AX/cells, decreased proliferation of cells, and an increased the percentage of G0/G1 phase cells and apoptotic cells. In vivo, the combination of oxaliplatin with PKI-587 inhibited tumor growth. Anti-tumor effects were associated with induction of mitochondrial apoptosis and inhibition of phosphorylation of mTOR, Akt and γ-H2AX. We conclude that PKI-587 enhances chemosensitivity of oxaliplatin in HCC through suppressing the PI3K/AKT/mTOR signalling pathway and inhibiting the DNA damage repair pathway. The combination of PKI-587 and oxaliplatin appears to be a promising regimen for the treatment of HCC.

BEZ235 enhances chemosensitivity of paclitaxel in hepatocellular carcinoma through inhibiting the PI3K/Akt/mTOR pathway.

Desensitization of hepatocellular carcinoma (HCC) to paclitaxel chemotherapy is a major deterrent to successful treatment of the cancer. Abnormal activation of the PI3K/Akt/mTOR, pathway is a common outcome of chemotherapy for HCC. Therefore, we investigated whether BEZ235, a dual PI3K and mTOR inhibitor, could increase the sensitivity of HCC to paclitaxel. In vitro results showed that paclitaxel, combined with BEZ235, inhibited HCC cell proliferation and migration, arrested the cell cycle in the G2/M phase, and promoted cell apoptosis by decreasing PI3K/Akt/mTOR activity. In vivo experiments confirmed that BEZ235 enhances the anti-tumor effect of paclitaxel by reducing PI3K/Akt/mTOR activity. Immunohistochemical staining showed that paclitaxel combined with BEZ235 reduced the numbers of Ki-67- and GPC3-positive HepG2 cells in tumor tissues. We conclude that BEZ235 enhanced the sensitivity of HCC to paclitaxel, and inhibition of PI3K/Akt/mTOR signaling might be a therapeutic strategy against paclitaxel-resistant HCC.