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In vitro evaluation of esomeprazole effect in head and neck squamous cell carcinomas

Matthew Nguyen, MD1; Li Zheng, DDS, PhD2; Petros Papagerakis, DDS, PhD3; Greg Wolf, MD1; Silvana Papagerakis, MD, PhD1

1KHRI, 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: mnewin@umich.edu

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.

References

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 10-7mM 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