Sustained EKR inhibition by EGFR targeting therapies is a predictive factor for synergistic cytotoxicity with PDT as neoadjuvant therapy

Background

Tyrosin kinase inhibitors (TKIs) and monoclonal antibodies aimed to target epidermal growth factor receptor (EGFR) have shown limited effect as monotherapies and drug resistance is a major limitation for therapeutic success. Adjuvant therapies to EGFR targeting therapeutics are therefore of high clinical relevance.

Methods

Three EGFR targeting drugs, Cetuximab, Erlotinib and Tyrphostin AG1478 were used in combination with photodynamic therapy (PDT) in two EGFR positive cell lines, A-431 epidermoid skin carcinoma and WiDr colorectal adenocarcinoma cells. The amphiphilic meso-tetraphenylporphine with 2 sulphonate groups on adjacent phenyl rings (TPPS_2a) was utilized as a photosensitizer for PDT. The cytotoxic outcome of the combined treatments was evaluated by cell counting and MTT. Cellular signalling was explored by Western blotting.

Results

PDT as neoadjuvant to Tyrphostin in A-431 cells as well as to Tyrphostin or Erlotinib in WiDr cells revealed synergistic cytotoxicity. In contrast, Erlotinib or Cetuximab combined with neoadjuvant PDT induced an antagonistic effect on cell survival of A-431 cells. Neoadjuvant PDT and EGFR targeting therapies induced a synergistic inhibition of ERK as well as synergistic cytotoxicity only when the EGFR targeting monotherapies caused a prolonged ERK inhibition. There were no correlation between EGFR inhibition by the EGFR targeting monotherapies or the combined therapies and the cytotoxic outcome combination-therapies.

Conclusions

The results suggest that sustained ERK inhibition by EGFR targeting monotherapies is a predictive factor for synergistic cytotoxicity when combined with neoadjuvant PDT.

General significance

The present study provides a rationale for selecting anticancer drugs which may benefit from PDT as adjuvant therapy.     

Extracellular ATP activates MAP kinase cascades through a P2Y purinergic receptor in the human intestinal Caco-2 cell line

Background

ATP exerts diverse effects on various cell types via specific purinergic P2Y receptors. Intracellular signaling cascades are the main routes of communication between P2Y receptors and regulatory targets in the cell.

Methods and results

We examined the role of ATP in the modulation of ERK1/2, JNK1/2, and p38 MAP kinases (MAPKs) in human colon cancer Caco-2 cells. Immunoblot analysis showed that ATP induces the phosphorylation of MAPKs in a time- and dose-dependent manner, peaking at 5 min at 10 µM ATP. Moreover, ATPγS, UTP, and UDP but not ADP or ADPβS increased phosphorylation of MAPKs, indicating the involvement of, at least, P2Y₂/P2Y₄ and P2Y₆ receptor subtypes. RT–PCR studies and PCR product sequencing supported the expression of P2Y₂ and P2Y₄ receptors in this cell line. Spectrofluorimetric measurements showed that cell stimulation with ATP induced transient elevations in intracellular calcium concentration. In addition, ATP-induced phosphorylation of MAPKs in Caco-2 cells was dependent on Src family tyrosine kinases, calcium influx, and intracellular Ca²⁺ release and was partially dependent on the cAMP/PKA and PKC pathways and the EGFR.

General significance

These findings provide new molecular basis for further understanding the mechanisms involved in ATP functions, as a signal transducer and activator of MAP kinase cascades, in colon adenocarcinoma Caco-2 cells.     

P2X7 receptor antagonists display agonist-like effects on cell signaling proteins

Background

The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca²⁺]_i. We report that some agents that can block P2X₇R receptors also promote diverse P2X₇R-independent effects on cell signaling.

Methods

We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X₇R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins.

Results

With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated ⁴⁵Ca²⁺ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X₇R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation.

Conclusions

Several agents used as P2X₇R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.

General significance

Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

Glycolysis–respiration relationships in a neuroblastoma cell line

Background

Although some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized.

Methods

We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions.

Results

Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased.

Conclusions

These data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration.

General significance

Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches.     

Berberine and S allyl cysteine mediated amelioration of DEN + CCl4 induced hepatocarcinoma

Background

Diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) have been used as initiator and promoter respectively to establish an animal model for investigating molecular events appear to be involved in development of liver cancer. Use of herbal medicine in therapeutics to avoid the recurrence of hepatocarcinoma has already generated considerable interest among oncologists. In this context studies involving S-allyl-cysteine (SAC) and berberine have come up with promising results. Here we have determined the individual effect of SAC and berberine on the biomolecules associated with DEN + CCl4 induced hepatocarcinoma. Effective therapeutic value of combined treatment has also been estimated.

Methods

ROS accumulation was analyzed by FACS following DCFDA incubation. Bcl2-Bax and HDAC1‐pMdm2 interaction were demonstrated by co-immunoprecipitation. Immunosorbent assay was performed to analyze PP2A and caspase3 activities. MMP was determined cytofluorimetrically by investigating JC-1 fluorescence. AnnexinV binding was demonstrated by labeling the cells with AnV-FITC followed by flow cytometry.

Results

CytochromeP4502E1 mediated bioactivation of DEN + CCl4 induced Akt dependent pMdm2‐HDAC1 interaction that led to p53 deacetylation, probable cause of its degradation. In parallel, oxidative stress dependent Nrf2‐HO1 activation increased Bcl2 expression which in turn stimulated cell proliferation. SAC in combination with berberine inhibited Akt mediated cell proliferation. Activation of PP2A as well as inhibition of JNK resulted in induction of apoptosis after 30 days of treatment. Extension of combined treatment reverted tissue physiology towards control. Co-treated group displayed normal tissue structure.

Conclusion and general significance

SAC and berberine mediated HDAC1/Akt inhibition implicates the efficacy of combined treatment in the amelioration of DEN + CCl4 induced hepatocarcinoma.     

Immune signal transduction in leishmaniasis from natural to artificial systems: Role of feedback loop insertion

Background

Modulated immune signal (CD14–TLR and TNF) in leishmaniasis can be linked to EGFR pathway involved in wound healing, through crosstalk points. This signaling network can be further linked to a synthetic gene circuit acting as a positive feedback loop to elicit a synchronized intercellular communication among the immune cells which may contribute to a better understanding of signaling dynamics in leishmaniasis.

Methods

Network reconstruction with positive feedback loop, simulation (ODE 15s solver) and sensitivity analysis of CD14–TLR, TNF and EGFR was done in SimBiology (MATLAB 7.11.1). Cytoscape and adjacency matrix were used to calculate network topology. PCA was extracted by using sensitivity coefficient in MATLAB. Model reduction was done using time, flux and sensitivity score.

Results

Network has five crosstalk points: NIK, IκB–NFκB and MKK (4/7, 3/6, 1/2) which show high flux and sensitivity. PI3K in EGFR pathway shows high flux and sensitivity. PCA score was high for cytoplasmic ERK1/2, PI3K, Atk, STAT1/3 and nuclear JNK. Of the 125 parameters, 20% are crucial as deduced by model reduction.

Conclusions

EGFR can be linked to CD14–TLR and TNF through the MAPK crosstalk points. These pathways may be controlled through Ras and Raf that lie upstream of signaling components ERK ½ (c) and JNK (n) that have a high PCA score via a synthetic gene circuit for activating cell–cell communication to elicit an inflammatory response. Also a disease resolving effect may be achieved through PI3K in the EGFR pathway.

General significance

The reconstructed signaling network can be linked to a gene circuit with a positive feedback loop, for cell–cell communication resulting in synchronized response in the immune cell population, for disease resolving effect in leishmaniasis.     

Circulating hormone adrenomedullin and its binding protein protect neural cells from hypoxia-induced apoptosis

Background

Brain ischemia is the underlying cause of neuron death during stroke and brain trauma. Neural cells exposed to ischemia can undergo apoptosis. Adrenomedullin (AM) in combination with its enhancing binding protein, AMBP-1, has been shown to reduce tissue damage in inflammation.

Methods

To evaluate a beneficial effect of AM/AMBP-1 administration in brain ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human neuroblastoma SH-SY5Y cells.

Results

After exposure to 1% O₂ for 20 h, neural cells were injured with decreased ATP levels and increased LDH release. Pre-administration of AM/AMBP-1 significantly reduced hypoxia-induced cell injury. Moreover, AM/AMBP-1 treatment reduced the number of TUNEL-positive cells and activation of caspase-3, compared to cells exposed to hypoxia alone. AM/AMBP-1 prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after hypoxia exposure. Correspondingly, an elevation of cAMP levels by forskolin protected neural cells from hypoxia-induced injury. Inhibition of PKA by KT5720 abolished the protective effect of AM/AMBP-1 on hypoxia-induced apoptosis.

Conclusions

AM/AMBP-1 elevates cAMP levels, followed by activating PKA, to protect neural cells from the injury caused by hypoxia.

General significance

AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from brain ischemia.     

COMP-Ang1 inhibits apoptosis as well as improves the attenuated osteogenic differentiation of mesenchymal stem cells induced by advanced glycation end products

Background

In the present study, we have investigated the possibility that cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), important factor in angiogenesis, osteogenesis and the survival of mesenchymal stem cells (MSCs) through the Ang1/Tie2 pathway has beneficial effects on osteogenic differentiated cells (ODCs) from MSCs treated by advanced glycation end products (AGE), which are pathological factors of diabetes.

Methods

Primary culture of MSCs was used. For comparison analysis of AGE and COMP-Ang1 effects, we performed cell viability assay with each treated variety concentration for 24 h. Apoptosis rate and Caspase-3 activity were measured by each ELISA assay. To make sure with Ang1/Tie2 pathway, we performed small interfering RNA transfected to MSCs. Real-time RT-PCR was performed to identify ODCs marker genes. Immunoblotting was used to evaluate the expression of Tie2, AKT, p38 and ERK.

Results

Our results clearly demonstrate that COMP-Ang1 upregulates the phosphorylation of AKT and p38 by activating the Ang1/Tie2 signaling pathway, indicating that COMP-Ang1 affects both AGE-induced apoptosis and the attenuated osteogenic differentiation of MSCs through the p38/MAPK and PI3K/AKT pathways.

Conclusions

COMP-Ang1 improves cell viability and differentiation function of ODCs against AGE via Ang/Tie2 signaling pathway.

General significance

Our results suggest the potential importance of COMP-Ang1 as a new therapy for impaired bone formation that is associated with diabetes and advanced age.     

Transducing properties of a pre-structured α-helical DPT-peptide containing a short canine adenovirus type 2 E4orf4 PP2A1-binding sequence

Background

Induction of the death pathway resulting from the specific interaction of the PP2A₁ phosphatase with adenoviral E4orf4 protein is a promising approach for cancer therapy. With the aim of deregulating tumor pathways, and mimicking E4orf4 anti-cancer signal, we have previously proposed the DPT technology concept, based on design of specific PP1/PP2A interacting penetrating peptides.

Methods

Using biochemical, structural and cell survival experiments, we have characterized new DPT-peptides containing short PP2A binding sequences.

Results

We identified overlapping sequences, located within the N-terminal domain E4orf4_23-46 of canine adenoviral E4orf4 protein, that interact with the PP2A-Bα subunit of PP2A₁ holoenzyme. We characterized DPT-E4orf4₄ and TAT-E4orf4₄, two bi-partite cell penetrating peptides containing the 12 PP2A₁ binding residues of the canine type 2 E4orf4_27-38 sequence, respectively fused to the DPT-sh1 and TAT shuttle sequences. Surprisingly DPT-E4orf4₄, in contrast to inactive TAT-E4orf4₄, adopted a well defined α-helical structure and co-precipitated PP2A₁ from HeLa cell extracts. DPT-E4orf4₄ also internalized streptavidin-HRP and inhibited survival of HeLa cells more efficiently than TAT, TAT-E4orf4₄ or the previously published anti-tumor TAT-derived peptide shepherdin. DPT-E4orf4₄ also efficiently inhibited the survival of human adherent transformed cells, including wild type and p53 mutated colonic HCT116 cells, without affecting survival of human non-transformed fibroblasts.

Conclusions

We characterized the transducing properties of a new α-helical DPT-E4orf4₄ peptide containing a short PP2A-interacting sequence from canine Adenoviral E4orf4 protein.

General significance

Our results suggest that α-helical structured DPT peptides specifically interacting with PP2A could be a valuable anti-cancer drug design scaffold.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

The activation of HO-1/Nrf-2 contributes to the protective effects of diallyl disulfide (DADS) against ethanol-induced oxidative stress

Background

Diallyl disulfide (DADS) is a garlic-derived organosulfur compound. The current study is designed to evaluate the protective effects of DADS against ethanol-induced oxidative stress, and to explore the underlying mechanisms by examining the HO-1/Nrf-2 pathway.

Methods

We investigated whether or not DADS could activate the HO-1 in normal human liver cell LO2, and then evaluated the protective effects of DADS against ethanol-induced damage in LO2 cells and in acute ethanol-intoxicated mice. The biochemical parameters were measured using commercial kits. HO-1 mRNA level was determined by RT-PCR. Histopathology and immunofluorescence assay were performed with routine methods. Protein levels were measured by western blot.

Results

DADS significantly increased the mRNA and protein levels of HO-1, stimulated the nuclear translocation of Nrf-2 and increased the phosphorylation of MAPK in LO2 cells. The nuclear translocation of Nrf-2 was abrogated by MAPK inhibitors. DADS significantly suppressed ethanol-induced elevation of lactate dehydrogenase (LDH) and aspartate transaminase (AST) activities, decrease of glutathione (GSH) level, increase of malondialdehyde (MDA) levels, and apoptosis of LO2 cells, which were all blocked by ZnPPIX. In mice, DADS effectively suppressed acute ethanol-induced elevation of aminotransferase activities, and improved liver histopathological changes, which might be associated with HO-1 activation.

Conclusion

These results demonstrate that DADS could induce the activation of HO-1/Nrf-2 pathway, which may contribute to the protective effects of DADS against ethanol-induced liver injury.

General significance

DADS may be beneficial for the prevention and treatment of ALD due to significant activation of HO-1/Nrf-2 pathway.     

Signaling related with biphasic effects of bisphenol A (BPA) on Sertoli cell proliferation: A comparative proteomic analysis

Background

Biphasic effects on cell proliferation of bisphenol A (BPA) can occur at lesser or greater exposures. Sertoli cells play a pivotal role in supporting proliferation and differentiation of germ cells. The mechanisms responsible for inverse effects of great and low concentrations of BPA on Sertoli cell proliferation need further study.

Methods

We utilized proteomic study to indentify the protein expression changes of Sertoli TM4 cells treated with 10^− 8 M and 10^− 5 M BPA. The further mechanisms related to mitochondria, energy metabolism and oxidative stress were investigated by qRT-PCR and Western-blotting analysis.

Results

Proteomic studies identified 36 proteins and two major clusters of proteins including energy metabolism and oxidative stress expressed with opposite changes in Sertoli cells treated with 10^− 8 M and 10^− 5 M BPA, respectively, for 24 h. Exposure to 10^− 5 M BPA resulted in greater oxidative stress and then inhibited cell proliferation, while ROS scavenger NAC effectively blocked these effects. Exposure to 10^− 8 M BPA caused higher intercellular ATP, greater activities of mitochondria, and resulted in significant proliferation of TM4 cells, while oligomycin A, an inhibitor of ATP synthase, abolished these growth advantages.

Conclusions

Our study demonstrated that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation by promoting energy metabolism.

General significance

Micromolar BPA inhibits cell proliferation by elevating oxidative stress while nanomolar BPA stimulates cell proliferation by promoting energy metabolism.     

CK2 phosphorylation of human Sec63 regulates its interaction with Sec62

Background

Protein kinase CK2 is a pleiotropic enzyme which is ubiquitously expressed in eukaryotic cells. Several years ago CK2 was found to be associated with the mammalian endoplasmic reticulum. So far nothing is known about the function of CK2 at the ER.

Methods

CK2 phosphorylation sites in the polypeptide chain of Sec63 were mapped using deletion mutants and a peptide library. Binding of Sec63 to CK2 and to Sec62 was analyzed by pull-down assays and by co-immunoprecipitation

Results

Sec63 was identified as a novel substrate and binding partner of protein kinase CK2.We identified serine 574, serine 576 and serine 748 as CK2 phosphorylation sites. Phosphorylation of Sec63 by CK2 enhanced its binding to Sec62.

Conclusions

Protein kinase CK2 phosphorylation of Sec63 leads to an enhanced binding of Sec63 to Sec62. This complex formation is a prerequisite for a functional ER protein translocon.

General significance

Thus, our present data indicate a regulatory role of CK2 in the ER protein translocation.