Asmaa El-Kenawi

Neutralization of Tumor Acidity Improves Antitumor Responses to Immunotherapy

Cancer immunotherapies, such as immune checkpoint blockade or adoptive T-cell transfer, can lead to durable responses in the clinic, but response rates remain low due to undefined suppression mechanisms. Solid tumors are characterized by a highly acidic microenvironment that might blunt the effectiveness of antitumor immunity. In this study, we directly investigated the effects of tumor acidity on the efficacy of immunotherapy. An acidic pH environment blocked T-cell activation and limited glycolysis in vitro. IFNγ release blocked by acidic pH did not occur at the level of steady-state mRNA, implying that the effect of acidity was posttranslational. Acidification did not affect cytoplasmic pH, suggesting that signals transduced by external acidity were likely mediated by specific acid-sensing receptors, four of which are expressed by T cells. Notably, neutralizing tumor acidity with bicarbonate monotherapy impaired the growth of some cancer types in mice where it was associated with increased T-cell infiltration. Furthermore, combining bicarbonate therapy with anti-CTLA-4, anti-PD1, or adoptive T-cell transfer improved antitumor responses in multiple models, including cures in some subjects. Overall, our findings show how raising intratumoral pH through oral buffers therapy can improve responses to immunotherapy, with the potential for immediate clinical translation.

Angiogenesis inhibitors in cancer therapy: mechanistic perspective on classification and treatment rationales

Angiogenesis, a process of new blood vessel formation, is a prerequisite for tumour growth to supply the proliferating tumour with oxygen and nutrients. The angiogenic process may contribute to tumour progression, invasion and metastasis, and is generally accepted as an indicator of tumour prognosis. Therefore, targeting tumour angiogenesis has become of high clinical relevance. The current review aimed to highlight mechanistic details of anti‐angiogenic therapies and how they relate to classification and treatment rationales. Angiogenesis inhibitors are classified into either direct inhibitors that target endothelial cells in the growing vasculature or indirect inhibitors that prevent the expression or block the activity of angiogenesis inducers. The latter class extends to include targeted therapy against oncogenes, conventional chemotherapeutic agents and drugs targeting other cells of the tumour micro‐environment. Angiogenesis inhibitors may be used as either monotherapy or in combination with other anticancer drugs. In this context, many preclinical and clinical studies revealed higher therapeutic effectiveness of the combined treatments compared with individual treatments. The proper understanding of synergistic treatment modalities of angiogenesis inhibitors as well as their wide range of cellular targets could provide effective tools for future therapies of many types of cancer.

Celecoxib, a Selective Cyclooxygenase-2 Inhibitor, Attenuates Renal Injury in a Rat Model of Cisplatin-Induced Nephrotoxicity

Background: Cisplatin is an effective chemotherapeutic agent successfully used in the treatment of a wide range of tumors. Nevertheless, nephrotoxicity has restricted its clinical use. Recent studies have strongly suggested that inflammatory mechanisms may play an important role in the pathogenesis of cisplatin nephrotoxicity. Celecoxib, a selective cyclooxygenase-2 inhibitor used as anti-inflammatory, may therefore have a protective effect on cisplatin-induced renal injury. Methods: In the present study, rats were injected intraperitoneally with a single dose of cisplatin (7 mg/kg) and/or celecoxib (30 mg/kg) for 5 days. Results: Nephrotoxicity manifested biochemically by elevations in serum creatinine, blood urea nitrogen, and proteinuria, and an increase in kidney weight as a percentage of total body weight. In addition, a marked decrease in serum albumin was observed. Lipid peroxidation in the kidney was monitored by measuring the malondialdehyde level and glutathione content, which were increased and depleted, respectively. Administration of celecoxib with cisplatin attenuated cisplatin-induced changes in kidney function parameters and oxidative stress markers. Histopathological examination of the kidney confirmed these results. Conclusion: In conclusion, this study indicates that celecoxib may be a promising drug for clinical use as a nephroprotectant against cisplatin-induced nephrotoxicity.

Protective effect of allicin against gentamicin-induced nephrotoxicity in rats

In this study, the modulator effect of allicin on the oxidative nephrotoxicity of gentamicin in the kidneys of rats was investigated by determining indices of lipid peroxidation and the activities of antioxidant enzymes, as well as by histological analyses. Furthermore, the effect of allicin on gentamicin induced hypersensitivity of urinary bladder rings to ACh was estimated. Twenty-four male Wistar albino rats were randomly divided into three groups, control, gentamicin (100mg/kg, i.p.) and gentamicin+allicin (50mg/kg, orally). At the end of the study, all rats were sacrificed and then urine, blood samples and kidneys were taken. Gentamicin administration caused a severe nephrotoxicity as evidenced by an elevated kidney/body weight ratio, serum creatinine, blood urea nitrogen (BUN), serum lactate dehydrogenase (LDH) and proteinuria with a reduction in serum albumin and creatinine clearance as compared with control group. In addition, a significant increase in renal contents of malondialdehyde (MDA), myeloperoxidase (MPO), nitric oxide (NOx) and tumor necrosis factor-alpha (TNF-α) concomitantly with a significant decrease in renal reduced glutathione (GSH) and superoxide dismutase (SOD) activities was detected upon gentamicin injection. Exposure to gentamicin increased the sensitivity of isolated urinary bladder rings to ACh and induced acute renal tubular epithelial cells necrosis. Administration of allicin significantly decreased kidney/body weight ratio, serum creatinine, LDH, renal MDA, MPO, NOx and TNF-α while it significantly increased creatinine clearance, renal GSH content and renal SOD activity when compared to gentamicin-treated group. Additionally, allicin significantly reduced the responses of isolated bladder rings to ACh and ameliorated tissue morphology as evidenced by histological evaluation. Our study indicates that allicin exerted protection against structural and functional damage induced by gentamicin possibly due to its antioxidant, anti-inflammatory and immunomodulatory properties in addition to its ability to retaining nitric oxide level.

Abstract 3213: Extracellular acidosis alters polarization of macrophages

Deficits in tumor perfusion and elevated glycolysis combine to reduce the pH of the tumor microenvironment. In poorly perfused volumes, oxygen and nutrient deprivation may also elicit necrotic areas that trigger infiltration of immune cells, including tumor-associated macrophages (TAMs). Inside tumors, TAMs are biased towards an M2 phenotype with anti-inflammatory and tumor-promoting characteristics, rather than an M1 phenotype known to be essential for host defense and tumor cell killing. We aimed to test whether low pH as a common stress factor in the tumor microenvironment could independently change the phenotypic characteristics and functional activity of macrophages. Accordingly, we stimulated macrophages into M1, M2 and prostate cancer-associated macrophages (Pr-TAMs) at pH 7.4 or pH 6.8 using IFN-γ+LPS, IL-4 or 30% TRAMP-C2-conditioned medium, respectively. 120 h after initiation of activation, M2 macrophages showed higher viability at pH 6.8 compared to pH 7.4, suggesting a better fitness of M2 macrophages in an acidic microenvironment. Extracellular acidosis also decreased gene expression of common pro-inflammatory M1 markers (iNOS, MCP1 and IL-6) in M1 macrophages; while it increased gene expression of M2 markers (MRC1, ARG1 and Chi313) in M2 macrophages. In Pr-TAMs, low pH decreased iNOS/ARG1 mRNA ratio indicating a shift towards the tumor promoting M2 phenotype. Using a cytokine array, release of inflammatory cytokines into media was also decreased when M1 macrophages were activated at acidic pH. Interestingly, when macrophages were incubated at neutral or low pH for 24 h then co-cultured with the prostate cancer cell lines TRAMP-C2 or -C3, the low pH- treated macrophages showed a higher mannose receptor expression compared to neutral pH-treated macrophages, suggesting a shifting towards M2 phenotype. Furthermore, TRAMP-C2 cells co-cultured with macrophages in acidic medium had a higher proliferation rate than tumor cells co-cultured at neutral pH. Notably, treating TRAMP mice with 200 mM sodium bicarbonate to raise microenvironmental pH, strongly inhibited both tumor progression and macrophage infiltration. In conclusion, extracellular acidosis induced a macrophage phenotypic switch on both gene expression and cytokine profile levels, which would lead to a promotion of tumor growth. Also, intervention of microenvironmental tumor acidity using buffer therapy in TRAMP mice decreased the number of tumor associated macrophages, coincident with retardation of tumor progression. Citation Format: Asmaa E. El-Kenawi, Arig A. Ibrahim-Hashim, Kimberly A. Luddy, Shari A. Pilon-Thomas, Robert A. Gatenby, Robert J. Gillies. Extracellular acidosis alters polarization of macrophages. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3213. doi:10.1158/1538-7445.AM2015-3213

Abstract 4560: Effects of cisplatin, vorinostat, and their combinations on EGFR-overexpressing cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cisplatin is one of the most commonly used drugs against human malignancies. Its cytotoxic effects are mainly mediated by formation of cisplatin-DNA adducts, which activate series of DNA damage-responsive proteins that ultimately trigger cell death. In spite of its potent activity, some malignancies treated with cisplatin may develop resistance after initial response while others may be intrinsically insensitive. Accordingly, cisplatin is frequently combined with other drugs to reduce resistance or to attain synergy. The presented work was conducted in EGFR-overexpressing cancer cells, DU-145 and MDA-MB-231, to examine the possible molecular effects of cisplatin alone or in combination with vorinostat, a histone deacetylase inhibitor. Proliferation assay, annexin V staining, western blotting and RT-PCR were used to study effects of those drugs on cell viability, apoptosis and molecular events in both cell lines. In the present study, incubation of DU-145 cells with cisplatin exerted dose-dependent antiproliferative and apoptosis-inducing effects. In addition, cisplatin significantly increased the apoptosis markers, cleaved PARP and cleaved caspase 7; depleted total EGFR and decreased ERK activation. This molecular response was also associated with dose-dependent increase in phosphorylated ATM (an early sensor of genotoxic stress), phosphorylated p38 and mRNA level of DDIT3 (a late sensor of DNA damage). On the other hand, in MDA-MB-231, cisplatin did not affect percent of apoptotic cells as compared with the untreated control, indicating a cisplatin-insensitive phenotype. The insensitive response towards cisplatin treatment was confirmed through absence of cleaved PARP and cleaved caspase 7 in total cell lysates. Although cisplatin increased the level of phosphorylated ATM and phosphorylated p38 in MDA-MB-231, it failed to affect DDIT3 mRNA level as compared with untreated cells. This may suggest that sensitivity towards cisplatin may be related to late DNA damage processing rather than early DNA damage response. Treating DU-145 and MDA-MB-231 cells with vorinostat elicited dose-dependent decrease in proliferation and increase in percentage of apoptotic cells. It also dose-dependently cleaved PARP and caspase 7, time-dependently degraded EGFR and decreased ERK activation in both cell lines. In spite of that potent activity, vorinostat synergized the effect of cisplatin only in DU-145 and failed to circumvent the insensitivity of MDA-MB-231 cells towards cisplatin. In conclusion, cisplatin decreased cell proliferation and induced apoptosis only in DU-145 cells but not MDA-MB-231 cells. Combination of vorinostat and cisplatin synergistically reduced percent cell viability and triggered apoptotic response in DU-145 cells when compared with the effects of individual drugs. However, in MDA-MB-231cells, addition of vorinostat to cisplatin did not significantly change any of the measured parameters. Citation Format: Asmaa Elsayed El-Kenawi, John Kenneth Cowell. Effects of cisplatin, vorinostat, and their combinations on EGFR-overexpressing cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4560. doi:10.1158/1538-7445.AM2014-4560

Combining radiomics and mathematical modeling to elucidate mechanisms of resistance to immune checkpoint blockade in non-small cell lung cancer

Immune therapies have shown promise in a number of cancers, and clinical trials using the anti-PD-L1/PD-1 checkpoint inhibitor in lung cancer have been successful for a number of patients. However, some patients either do not respond to the treatment or have cancer recurrence after an initial response. It is not clear which patients might fall into these categories or what mechanisms are responsible for treatment failure. To explore the different underlying biological mechanisms of resistance, we created a spatially explicit mathematical model with a modular framework. This construction enables different potential mechanisms to be turned on and off in order to adjust specific tumor and tissue interactions to match a specific patients disease. In parallel, we developed a software suite to identify significant computed tomography (CT) imaging features correlated with outcome using data from an anti-PDL-1 checkpoint inhibitor clinical trial for lung cancer and a tool that extracts these features from both patient CT images and “virtual CT” images created from the cellular density profile of the model. The combination of our two toolkits provides a framework that feeds patient data through an iterative pipeline to identify predictive imaging features associated with outcome, whilst at the same time proposing hypotheses about the underlying resistance mechanisms.

Allicin ameliorates kidney function and urinary bladder sensitivity in cyclosporine A-treated rats.

Cyclosporine-A (CsA) is an immunosuppressive drug which has been used to prevent rejection after organ transplantation and to treat certain autoimmune diseases. However, its therapeutic use is limited by nephrotoxicity. In this study, the modulator effect of allicin on the oxidative nephrotoxicity of CsA in rats was investigated. Furthermore, the effect of allicin on CsA-induced hypersensitivity of urinary bladder rings to acetylcholine (ACh) was estimated. Rats were divided into three groups, control, CsA (15 mg/kg, subcutaneously), and CsA/allicin (50 mg/kg, orally). At the end of the study, all rats were killed and then blood, urine samples, and kidneys were taken. CsA administration caused a severe nephrotoxicity which was evidenced by elevated kidney/body weight ratio, serum creatinine (Cr), blood urea nitrogen, lactate dehydrogenase, and urinary protein with a concomitant reduction in serum albumin and Cr clearance as compared with control. A significant increase in renal contents of malondialdehyde, myeloperoxidase, and tumor necrosis factor-alpha with a significant decrease in renal reduced glutathione, superoxide dismutase activities, and nitric oxide (NOx) content was detected upon CsA administration. Exposure to CsA increased the sensitivity of isolated urinary bladder rings to ACh. Histological analysis revealed that CsA caused tubular necrosis and moderate diffuse tubular atrophy. Allicin protected kidney tissue against the oxidative damage and the nephrotoxic effect of CsA and significantly reduced the responses of isolated bladder rings to ACh. Our study indicates that allicin administration has the potential to protect against CsA-induced renal injury by reducing oxidative stress and inflammation and restoring NOx level.

Abstract 1029: Involvement of endocytic pathway in vorinostat-induced EGFR degradation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Epidermal growth factor receptor (EGFR) is frequently overexpressed or aberrantly activated in various malignancies. As a result, EGFR is considered an important target in cancer therapeutics. Strategies of EGFR targeting include blockade of its activation, inhibition of downstream signaling or induction of EGFR downregulation and/or degradation. Histone deacetylases (HDACs) inhibitors were previously shown to deplete EGFR through numerous mechanisms varying from suppression of EGFR transcription, attenuation of its mRNA stability and disruption of chaperone activity of HSP90. In this work, we are studying alteration of EGFR vesicular trafficking as another mechanism that may be involved in HDAC inhibitors-induced EGFR depletion. Cytotoxic and EGFR-degrading effects of vorinostat, a pan-HDAC inhibitor, were investigated in the EGFR-overexpressing cell lines, DU-145 (prostate cancer cells) and MDA-MB-231 (breast cancer cells). Using different experimental designs, dose- and time- effects of vorinostat were studied in both cell lines. Proliferation assay, annexin V staining, PARP and caspase 7 cleavage were used to evaluate overall cellular toxicity. Effects on total EGFR protein level and relevant ERK phosphorylation were also investigated by immunoblotting. Moreover, accumulation of acetyl-α-tubulin was verified through immunoblotting and immunofluorescence imaging. Finally, serum-starved vorinostat-treated cells were stimulated with EGF in order to elucidate effect of vorinostat on EGF-induced EGFR degradation, an endocytic trafficking-dependent process. As presented in this study, vorinostat exerted potent cytotoxic effects on DU-145 and MDA-MB-231 cells manifested by a dose-dependent decrease in proliferation and increase in percentage of apoptotic cells. In addition, vorinostat caused a dose-dependent cleavage of both PARP and caspase 7, two markers of apoptotic cascade activation. Furthermore, vorinostat elicited a dose- and a time-dependent depletion of EGFR total protein in both cell lines with a corresponding decrease in ERK activation. Add to that, vorinostat caused accumulation of acetyl-α-tubulin and promoted EGFR degradation upon EGF stimulation denoting accelerated endocytic trafficking. In conclusion, vorinostat demonstrated a potent anti-tumor activity against EGFR-overexpressing cell lines. Such activity was accompanied by EGFR depletion that may be contributed in part by vorinostat-induced deregulated vesicular trafficking.  Citation Format: Asmaa E. Elkenawi, John K. Cowell. Involvement of endocytic pathway in vorinostat-induced EGFR degradation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1029. doi:10.1158/1538-7445.AM2013-1029