Constitutive Cell Proliferation Regulating Inhibitor of Protein Phosphatase 2A (CIP2A) Mediates Drug Resistance to Erlotinib in an EGFR Activating Mutated NSCLC Cell Line

Exploring mechanisms of drug resistance to targeted small molecule drugs is critical for an extended clinical benefit in the treatment of non-small cell lung cancer (NSCLC) patients carrying activating epidermal growth factor receptor (EGFR) mutations. Here, we identified constitutive cell proliferation regulating inhibitor of protein phosphatase 2A (CIP2A) in the HCC4006rErlo0.5 NSCLC cell line adapted to erlotinib as a model of acquired drug resistance. Constitutive CIP2A resulted in a constitutive activation of Akt signaling. The proteasome inhibitor bortezomib was able to reduce CIP2A levels, which resulted in an activation of protein phosphatase 2A and deactivation of Akt. Combination experiments with erlotinib and bortezomib revealed a lack of interaction between the two drugs. However, the effect size of bortezomib was higher in HCC4006rErlo0.5, compared to the erlotinib-sensitive HCC4006 cells, as indicated by an increase in Emax (0.911 (95%CI 0.867–0.954) vs. 0.585 (95%CI 0.568–0.622), respectively) and decrease in EC50 (52.4 µM (95%CI 46.1–58.8 µM) vs. 73.0 µM (95%CI 60.4–111 µM), respectively) in the concentration–effect model, an earlier onset of cell death induction, and a reduced colony surviving fraction (0.38 ± 0.18 vs. 0.95 ± 0.25, respectively, n = 3, p < 0.05). Therefore, modulation of CIP2A with bortezomib could be an interesting approach to overcome drug resistance to erlotinib treatment in NSC

Cisplatin resistance in non-small cell lung cancer cells is associated with an abrogation of cisplatin-induced G2/M cell cycle arrest

The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 with regard to cisplatin resistance mechanisms including cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA damage response. In A549rCDDP2000 cells, a cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was reduced compared to A549 cells, although equitoxic cisplatin concentrations resulted in comparable platinum-DNA adduct levels. These differences were accompanied by changes in the expression of proteins involved in DNA damage response. In A549 cells, cisplatin exposure led to a significantly higher expression of genes coding for proteins mediating G2/M arrest and apoptosis (mouse double minute 2 homolog (MDM2), xeroderma pigmentosum complementation group C (XPC), stress inducible protein (SIP) and p21) compared to resistant cells. This was underlined by significantly higher protein levels of phosphorylated Ataxia telangiectasia mutated (pAtm) and p53 in A549 cells compared to their respective untreated control. The results were compiled in a preliminary model of resistance-associated signaling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G2/M cell cycle arrest and apoptosis

Assessment of Serum Vitamin D in Patients with Bronchial Asthma

Background: Asthma is one of the most common chronic diseases worldwide and has been increasing in prevalence over the last few decades; its exact cause remains unknown and likely has its origin in complex interactions among multiple genetic and environmental factors. The World Health Organization (WHO) stated that approximately 300 million people have asthma. Recently the effects of vitamin D as a hormone have gained attention. Vitamin D appears to have regulatory effects on every part of the immune system, vitamin D deficiency being linked to an array of immunologically based diseases focusing on asthma. So the assessment of serum vitamin D in patients with bronchial asthma is useful. Objective: The aim of this study is to assess the level of serum vitamin D in patients with bronchial asthma. Methods: The present study, included 50 subjects, 40 patients diagnosed as bronchial asthma and 10 healthy volunteers. The asthmatic patient group was divided into group (A) asthma in between attack and group (B) exacerbated asthma, that was divided into subgroups (I), (II) and (III) mild, moderate and severe exacerbated asthma, respectively according to severity of symptoms and FEV1. Results: Serum vitamin D level was highly significantly lower in the asthmatic patient group compared to the control subject group and lowest serum 25 hydroxy(OH) vitamin D value in the severely exacerbated asthma group. There was a positive correlation between decreased serum 25 (OH)D levels and decreased FEV1. Conclusions: Vitamin D deficiency was highly prevalent in asthmatic patients, there was a strong correlation between asthma severity and 25(OH) vitamin D concentrations and there was a direct and a positive significant correlation between vitamin D levels and pulmonary function tests in asthmatic patients, so the measurement of serum vitamin D levels in patients with bronchial asthma is very useful

Assessment of PULP score in predicting 30-day perforated duodenal ulcer morbidity, and comparison of its performance with Boey and ASA, a retrospective study

Background: /aim: Scores commonly employed to risk stratify perforated peptic ulcer patients include ASA (American Society of Anesthesiologists), Boey and peptic ulcer perforation score (PULP). However, few studies assessed and compared the accuracy indices of these three scores in predicting post PPU repair 30-day morbidity. We assessed accuracy indices of PULP, and compared them to Boey and ASA in predicting post perforated duodenal (PDU) ulcer repair 30-day morbidity. Methods: Retrospective chart review of all PDU patients (perforated duodenal ulcers only) at the largest two hospitals in Qatar (N = 152). Data included demographic, clinical, laboratory, operative, and post repair 30-day morbidity. Area under the Curve (AUC), sensitivity and specificity were computed for each of the 3 scores. Multivariate logistic regression assessed the accuracy indices of each score. Results: All patients were males (M age 37.41 years). Post PDU repair 30-day morbidity was 10.5% (16 morbidities). Older age, higher ASA (≥3), Boey (≥1) or PULP (≥8) scores, shock on admission and preoperative comorbidities; and conversely, lower hemoglobin and albumin were all positively significantly associated with higher post PDU 30-day morbidity. PULP displayed the largest AUC (72%), and was the only score to significantly predict 30-day morbidity. The current study is the first to report the sensitivity and specificity of these three scores for post PDU repair 30-day morbidity; and first to assess accuracy indices for PULP in predicting post PDU repair 30-day morbidity. Conclusion: PULP score had the largest AUC and was the only score to significantly predict post PDU repair 30-day morbidity. © 2019 The Author(s

Utilising the EGFR interactome to identify mechanisms of drug resistance in non-small cell lung cancer - Proof of concept towards a systems pharmacology approach

Drug treatment of epidermal growth factor receptor (EGFR) positive non-small cell lung cancer has improved substantially by targeting activating mutations within the receptor tyrosine kinase domain. However, the development of drug resistance still limits this approach. As root causes, large heterogeneity between tumour entities but also within tumour cells have been suggested. Therefore, approaches to identify these multitude and complex mechanisms are urgently required. Affinity purification coupled with high resolution mass spectrometry was applied to isolate and characterise the EGFR interactome from HCC4006 non-small cell lung cancer cells and their variant HCC4006(r)ERLO(0.5) adapted to grow in the presence of therapeutically relevant concentrations of erlotinib. Bioinformatics analyses were carried out to identify proteins and their related molecular functions that interact differentially with EGFR in the untreated state or when incubated with erlotinib prior to EGFR activation. Across all experimental conditions 375 proteins were detected to participate in the EGFR interactome, 90% of which constituted a complex protein interaction network that was bioinformatically reconstructed from literature data. Treatment of HCC4006(r)ERLO(0.5) cells carrying a resistance phenotype to erlotinib was associated with an increase of protein levels of members of the clathrin-associated adaptor protein family AP2 (AP2A1, AP2A2, AP2B1), structural proteins of cytoskeleton rearrangement as well as signalling molecules such as Shc. Validation experiments confirmed activation of the Ras-Raf-Mek-Erk (MAPK)-pathway, of which Shc is an initiating adaptor molecule, in HCC4006(r)ERLO(0.5) cells. Taken together, differential proteins in the EGFR interactome of HCC4006(r)ERLO(0.5) cells were identified that could be related to multiple resistance mechanisms including alterations in growth factor receptor expression, cellular remodelling processes suggesting epithelial-to-mesenchymal transition as well as alterations in downstream signalling. Knowledge of these mechanisms is a pivotal step to build an integrative model of drug resistance in a systems pharmacology manner and to be able to investigate the interplay of these mechanisms and ultimately recommend combinatorial treatment strategies to overcome drug resistance

Cisplatin resistance in non-small cell lung cancer cells is associated with an abrogation of cisplatin-induced G2/M cell cycle arrest

The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 with regard to cisplatin resistance mechanisms including cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA damage response. In A549rCDDP2000 cells, a cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was reduced compared to A549 cells, although equitoxic cisplatin concentrations resulted in comparable platinum-DNA adduct levels. These differences were accompanied by changes in the expression of proteins involved in DNA damage response. In A549 cells, cisplatin exposure led to a significantly higher expression of genes coding for proteins mediating G2/M arrest and apoptosis (mouse double minute 2 homolog (MDM2), xeroderma pigmentosum complementation group C (XPC), stress inducible protein (SIP) and p21) compared to resistant cells. This was underlined by significantly higher protein levels of phosphorylated Ataxia telangiectasia mutated (pAtm) and p53 in A549 cells compared to their respective untreated control. The results were compiled in a preliminary model of resistance-associated signaling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G2/M cell cycle arrest and apoptosis

2017 CERN-Fermilab HCP Summer School

<p>Analysis of XPC in a) RT-PCR (n = 3) and b) Western Blot (n = 3) as fold change relative to untreated control in A549 and A549^rCDDP²⁰⁰⁰ cells, presented as mean ± SEM with c) one representative western blot showing A549 untreated, A549 treated with 11 μM cisplatin, A549^rCDDP²⁰⁰⁰ untreated, A549^rCDDP²⁰⁰⁰ treated with 11 μM cisplatin and A549^rCDDP²⁰⁰⁰ treated with 34 μM cisplatin.</p

p21 mRNA and protein analysis.

<p>Analysis of p21 in a) RT-PCR (n = 3) and b) Western Blot (n = 3) as fold change relative to untreated control in A549 and A549^rCDDP²⁰⁰⁰ cells, presented as mean ± SEM with c) one representative western blot showing A549 untreated, A549 treated with 11 μM cisplatin, A549^rCDDP²⁰⁰⁰ untreated, A549^rCDDP²⁰⁰⁰ treated with 11 μM cisplatin and A549^rCDDP²⁰⁰⁰ treated with 34 μM cisplatin.</p

SIP mRNA and protein analysis.

<p>Analysis of SIP in a) RT-PCR (n = 3) and b) Western Blot (n = 3) as fold change relative to untreated control in A549 and A549^rCDDP²⁰⁰⁰ cells, presented as mean ± SEM with c) one representative western blot showing A549 untreated, A549 treated with 11 μM cisplatin, A549^rCDDP²⁰⁰⁰ untreated, A549^rCDDP²⁰⁰⁰ treated with 11 μM cisplatin and A549^rCDDP²⁰⁰⁰ treated with 34 μM cisplatin.</p

Primer and probe sequences for RT-PCR.

<p>Primer and probe sequences for RT-PCR.</p