In vitro evaluation of esomeprazole effect in head and neck squamous cell carcinomas Matthew Nguyen, MD 1 ; Li Zheng, DDS, PhD 2 ; Petros Papagerakis, DDS, PhD 3 ; Greg Wolf, MD 1 ; Silvana Papagerakis, MD, PhD 1 1 KHRI, Otolaryngology; 2 Otolaryngology and Pediatric Dentistry; 3 Pediatric Dentistry and Orthodontics University of Michigan, Ann Arbor, USA Matt Ng University of Michigan, Ann Arbor, MI 48109 MSRB 3, #9323 Email: [email protected] Contact 1. Papagerakis S, Bellile E, Peterson LA. Proton pump inhibitors and histamine 2 blockers are associated with improved overall survival in patients with head and neck squamous carcinoma. Cancer Prev Res. 2014 Dec;7(12):1258-69. 2. BCECF. https://tools.thermofisher.com/content/sfs/manuals/mp01150.pdf 3. Double immunofluorescence. http://www.abcam.com/ps/pdf/protocols/double%20immunofluorescence%20- simultaneous%20protocol.pdf 4. Nijkamp MM, Span PN, Hoogsteen IJ. Expression of E-cadherin and Vimentin correlates with metastasis formation in head and neck squamous cell carcinoma patients. Radiother Oncol. 2011 Jun;99(3):344-8. 5. Fais S, DeMilito A, You H Targeting Vacuolar H + -ATPases as a New Strategy against Cancer. Cancer Res. 2015 Nov;2007 67; 10627 6. Umemura N, Zhu J, Mburu YK. Defective NF-κB signaling in metastatic head and neck cancer cells leads to enhanced apoptosis by double-stranded RNA. Cancer Res. 2012 Jan 1;72(1):45-55. Cancer is the second most common killer in developed countries with increasing number of aging population. In US, there are an estimated 640,000 new cases of HNSCC diagnosed annually. Patients with local advanced head and cancer are at risk of recurrence and metastasis. The standard of care including combined chemo- radiation therapies with cisplatin and 5-FU or paclitaxel rendered response rates ranging from 30% to 40%, with median survival of 6 to 9 months. In less fortunate situations, metastasis or invasive recurrence develop, unresectable, or unresponsive to conventional treatments. At the University of Michigan, proton pump inhibitor (PPI) medications are commonly and regularly administered in patients with HNSCC as part of their cancer treatment for the management of acid reflux and gastric disturbance from conventional therapies. Recent studies completed at the University of Michigan have identified a significant association of the proton pump inhibitor (PPI) class of PPI class drugs with treatment outcomes and survival in patients with HNSCC; in addition when considering PPI drugs individually, the association with patient overall survival was maintained for esomeprazole (p=0.001) 1 . There are several hypothesis on how PPI play the role in modifying cancer cells both directly and in directly. Of those, modification of microenvironment via V-ATPase proton pump, epithelial-mesenchymal transformation as well as signaling pathway (NF-kB) are specifically emerging and drawing much of the interest. Background Cell lines: Cell line usage was regulated by the protocol approval of Institutional Review Board of University of Michigan: UMSCC-103, primary lateral tongue stage IV. Cell culture: Cells were cultured in adherent flasks (Corning, BDscience, USA ), DMEM (Gibco, NY, USA) supplemented with 100 IU/mL penicillin-streptomycin (Invitrogen, Carlsbad, CA) and 10% certified fetal bovine serum (Life Technologies, NY, USA) in the mycoplasma-free humidified incubator at 37oC, 5% CO2. Cells grow to 50-60% confluence and passage at 1:4 ratio. In vitro treatment with proton pump inhibitor, (IC50) determination* Esomeprazole (Abcam, Massachusetts, US) was dissolved in sterile 0.9% normal saline at concentration of 10mM. The dosage was established by IC50 determination assay using CCK-8 (WST) cell proliferation kit (Dojindo, Maryland, US). Cells were plated in 96-well plate (Corning, US) at a density of 10,000 cells/well in triplicate, treated with esomeprazole in 24 hours, then incubated with WST for 2 hours. The absorbance curve was plotted with a series of treating concentration exponentially increasing from 10 -7 mM to 10mM. IC50 was calculated at 50% reduction in absorbance rate of WST, equivalent to 50% cell population reduction. Proliferation assay*: Cells were plated in 90-well plate (Corning, US) at a density of 2000 cells/well in quadruplicate, treated with esomeprazole at the IC50 concentration (determined by the above assay). CCK-8 kit was used to monitor the cell survival and proliferation every 24 hours. *Absorbance rates were read by Cytation 3 microplate reader (Biotek, Winooski, Vermont, US) at 450 nm wavelength. Wound assay: Cells were plate in 24-well plate (Corning, US) in triplicate and allowed to grow up to 90% confluence. Cell monolayer was then scratched using 200uL pipette tip at midline of each well and allowed to grow in the incubator. Gap closure was monitored every 12 hours and 24 hours for UMSCC-103 and UMSCC-14A, 14B, respectively, as UMSCC-103 being a fast grower, under Nikon inverted microscope at 100x magnification and captured using Nikon microscope camera and software (Nikon, USA). The gap area was masked and measured by T-Scratch (Zurich, Switzerland). The subsequent plotting and t-test were performed by GraphPad Prism. Materials and Methods These findings lend additional support to the role of PPI in modifying the tumor microenvironment that held promise towards new therapeutic and prevention approaches with agents with minimal toxicities. Conclusions Objective Objective: Evaluate the effectiveness of esomeprazole on UMSCC-103 cell line at gene and protein levels. 1. Determine IC50 of esomeprazole. Evaluate inhibitory effect with proliferation assay and wound assay. 2. Evaluate the modification of tumor cells’ microenvironment pH with intracellular pH measurement and V-ATPase pump expression level. 3. Evaluate the effect on the cancer cell epithelial-mesenchymal transformation (EMT) with 2 representative markers: E-Cadherin (epithelial) and Vimentin (mesenchymal). 4. Evaluate the effect on cell NF-kB pathway. Our data indicates that esomeprazole has the ability to inhibit cancer cell proliferation and to influence epithelial-mesenchymal transformation in HNSCC with reduction of Vimentin filament and increase of E-Cadherin which promotes cell-cell adhesion, potentially prevent the cell break away and metastasis 4 . Low pH microenvironment around the tumors has been considered optimal condition for activation of proteases include matrix metalloproteinases (MMPs), bone morphogenetic protein-1-type metalloproteinases, tissue serine proteases and adamalysin-related membrane proteases 5 . This activation will help the cell break away from its origin and metastasize. By inhibiting the V-ATPase, esomeprazole alters transporting of hydrogen ion across cancer cell membrane, prevents metastatic invasion and dissemination. NF-kB is a protein regulating the DNA transcription, promoting cell proliferation and cell survival, inhibiting apoptosis 6 . NF-kB level is 1.46-fold lower in treated group. By altering the NK-kB levels, esomeprazole makes the cell become more sensitive to the action of antineoplastic agents. Discussion Figure 1. Half minimum inhibitory concentration IC50 of esomeprazole on UMSCC-103 was determined to be 112uM. Figure 2. Esomeprazole shows inhibitory effects in Proliferation assay (A) and Wound assay (B) Figure 3. Esomeprazole inhibits the V- ATPase on the cell membrane, preventing hydrogen ion pumped out of the cells, eventually lowering the intracellular pH. We noticed that the pHi in treated group were consistent over the quadruplicate (shown with shorter error bars), also, pHi was lowered in treated group but not decreased to acidic range. Our speculation is that esomeprazole lowers and stabilizes the pHi. However, there are several other pumps that also move the hydrogen across the membrane play the role in compensating the V- ATPase inhibition of hydrogen ion exchange. Figure 4. IF has been shown the expression of E-Cadherin, an epithelial marker increased in treatment group. The expression of Vimentin, a mesenchymal marker, decreased in esomeprazole-treated group. It is clearly noted under microscope that Vimentin filament are disrupted, clumped together in treatment group. A B Figure 5. On real time PCR, E-Cadherin was expressed 3-fold higher in treatment group. Vimentin was 1.3-fold higher in treatment group, this is discordance with IF quantification. We speculate that by disrupting the Vimentin, cancer cells are up-regulating the Vimentin production at gene level. ATP6V1C1 expressed 2.6 times higher. We also noted that the NF- kB was 1.46-fold less expressed in treatment group. Figure 6. Western Blot result shows V-ATPase protein levels were expressed less in treatment group. As a result, cells are up-regulating the production of V-ATPase at gene level (as shown in real time PCR result above). Intracellular pH measurement: 2’,7’-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF- AM, B3051) (Invitrogen, US) was utilized to measure the intracellular pH. Cells were plated in 96-well plate at a density of 3000 cells/well, in quadruplicate and treated with esomeprazole at IC50 concentration. At 24 hour interval, cells were incubated with 1uM BCECF for 30 minutes at 37oC. Fluorescence intensities were measured at 535nm with excitations at 440nm and 490nm wavelengths. The pH were calculated by the equation as described in the BCECF guide (Invitrogen) 2 . Immunofluorescence: Cells were plate on a sterile square cover slip in 6-well plate (Corning, US), and treated with esomeprazole at IC50 concentration. Staining steps were followed the standard protocol (Abcam) 3 . Antibody being used were E-Cadherin (3195, Cell Signaling Technology, Danvers, Massachusetts, US) at dilution 1:50 and Vimentin (V9, sc-6260, Santa Cruz, California, US) at 1:100 dilution. Immunofluorescence image was obtained with Nikon A1 confocal microscope at 100x magnification. Relative intensity and cell size were semi-quantified by Image J software (NIH, Bethesda, Maryland, US). RNA isolation and real time PCR. Cells were lysed by b-mercaptoethanol in Buffer RLT and isolated using Spin Technology and Qiagen kit (Qiagen, Valencia, CA). 2ug RNA product were then reverse transcribed with TaqMan reverse transcription reagents (Applied Biosystems, Branchbury, NJ, USA). The subsequent cDNA product was amplified by real time PCR using AmpliTaq Gold DNA polymerase (Applied Biosystems, Branchbury, NJ, USA) at 95oC for 30 seconds, 60oC for 30 seconds and 72oC for 30 seconds with specific primers for the genes of interest and GAPDH being the house-keeping gene. Western Blot: Cells are washed twice with PBS before being lysed for 20 min on ice in RIPA lysis buffer. Total proteins (20 μg) per lane are separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) using semidry method. The membrane is blocked with 5% nonfat milk, incubated overnight at 4°C with the primary antibody against V-ATPase (1:500, Sigma). Anti-GAPDH antibody (1:1000, Chemicon) is used to determine the loading control. The day after, the membranes are incubated for 1h at room temperature with horseradish peroxidase-conjugated (secondary) antibodies (1:4000, GE, NA931 and NA934), after washing. Bound antibodies are visualized by enhanced chemiluminescence (ECL) detection system. All bands are measured by densitometry and normalized to GAPDH (means±standard error of three measurements) using the ImageJ. Materials and Methods This study has been supported by the American Cancer Society RSG-13-103-01- CCE (SP) and NIDCD T32DC005356 (MN). Acknowledgements References