“Comparative Study of Salivary-Derived Protease Activity for the Early Diagnosis of Oral Squamous Cell Carcinoma”

Alberto Palacios-Carbajal1, Timothy Griffin Ph.D.2, Ebbing De Jong Ph.D.2

1University of California, San Diego; 2University of Minnesota, College of Biological Sciences: Department of Biochemistry, Molecular Biology and Biophysics ABSTRACT

Oral Squamous Cell Carcinoma (OSCC) is the sixth most common cancer and it has a five-year survival rate of 50%; while it could be 90% by early diagnosis. Studies suggest saliva to be a viable source for disease screening because of its composition that originates from salivary glands, serum and exfoliated cells (and because saliva bathes oral tumors). Comparative studies of healthy and OSCC patients have shown significant differences in salivary pH, salts, proteins and enzyme concentration. Also, in OSSC tissue, high levels of proteases have been implicated in metastatic spread. Based on these observations, our hypothesis was that OSCC may be detected at early stages by differences in salivary protease activity. To test our assumption, saliva was collected and presented with the nonspecific substrate, azocasein, to yield a colorimetric analysis by absorbance at 450 nm. Results showed significant protease activity in both healthy saliva supernatant and from the cellular portion. Also, proteases in Whole Saliva activity was maintained after several thaw cycles—that are common to clinical saliva expediting. Comparison of saliva from healthy and OSCC patients showed a trend where OSCC sample had a higher pH value, however no significant difference in protease activity was observed. Finally, early detection of OCSS via noninvasive salivary assay may have profound implications.

INTRODUCTIONOral Squamous Cell Carcinoma (OSCC) is a

common malignant tumor that makes 90% of all oral cancers with an increasing incidence and with the 50% five-year survival rate unchanged for at least two decades1,2. Currently, detection of OSCC is based on expert clinical examination and histological analysis of biopsied-premalignant areas. Inherently, these methods are flawed and /or impracticable. Therefore, a new approach is in demand.

Saliva has been said to mirror human’s internal health because components originate from serum and exfoliated cells. In OSCC patient’s saliva and tissue, an increase of total protein and proteases has been found; some of which are believed to play a significant general role tumor cell invasion, growth, and transformation. Some proteins being, nicotinamide N –methyltransferase (NNMT),2 matrix metalloproteinase (MMP) and insulin growth factors (IGF).3

Base on the scope of OSCC and the impetus of proteases in physiological pathology, Dr. Timothy J. Griffin and Dr. Ebbing De Jong aim to study oral cancer progression and the salivary proteome using a variety of quantitative proteomic strategies. Thus, the study that follows may represent a simple, cheap, and non-invasive approach that may have profound implications in the early detection of OSCC.

RESULTS Saliva supernatant buffered at pH 7.85 with 60ul of

1M Tris yield the optimal conditions for protease activity in the presence of azocasein-substrate (Fig.1). Comparison of OSCC and healthy saliva supernatant was done in the presence of buffer (pH 7.85) where the average proteolytic activity absorbance was 0.087 and 0.0823 (Fig.2B). Also, a pH trend was observed (and noted by the literature3) where OSCC saliva was more basic than healthy samples, pH 6.85 & 7.02 respectively (Fig.2A). In addition, Protease activity of saliva sample subjected to three freeze-thaw (-70Co) cycles maintained an average a of 0.103 (Fig. 3A) indicating saliva’s protease integrity to have been preserved. Lastly, preliminary experiments of cell lysis with sonicator increased the absorbance suggesting a liberation of proteases (Fig 3B).

CONCLUSIONSSaliva has potential in early detection and in monitoring disease development of OSCC because of the advantages in accessibility, low invasive procedure and low cost. In this study, saliva was shown to have detectable protease activity using a simple assay test and under various conditions including the common freeze storage procedure. However, it may be of interest to explore the comparison of protease activity of OSCC and healthy lysed cells. Also, studies of salivary proteases by Enzyme-Linked Immunosorbent Assay (ELISA) and studies of protease-substrate specificity may provide other venues in early OSCC detection.

ACKNOWLEDGMENTSCollege of Biological Sciences, U. Of Minnesota: Timothy J. Griffin Ph.D., Ebbing de Jong Ph.D. Life Science Summer Undergraduate Research

Program (LSSURP): Adrienne Watson Ph.D, Jon Gottesman Ph.D.,

Evelyn Juliussen Sponsored by NSF-REU in Molecular Genetics &

Proteomics

REFERENCES1.Joel A. Kooren, Nelson L. Rhodus, Timothy Griffin. “Emerging Non-Invasively Collected Genomic and Proteomic Biomarkers for the Early Diagnosis of Oral Squamous Cell Carcinoma (OSCC).” Squamous Cell Carcinoma2. Sartini, Davide et. al., “Analysis of Tissue and Saliva Nicotinamide N-methyltransferase in Oral Squamous Cell Carcinoma: Basis for Development of a Noninvasive Diagnostic Test for Early-Stage Disease.” Bio. Chem. (6/2012) vol. 393: 505-511 3. Thomas Shpitzer et al. “A comprehensive salivary analysis for oral cancer diagnosis.” J Cancer Res Clin Oncol (2007) 133:613–617•Eben L. Rosenthal, MD et al., “Expression of Proteolytic Enzymes in Head and NeckCancer–Associated Fibroblasts.” Arch Otolaryngol Head and Neck Surg (2004) 130: 943-47•Jesse Charney and Rudolph M. Tomarelli. “Duodental Juice Proteolytic Activity of Determination of the Colorimetric Method…”J. Biol. Chem. 1947, 171:501-505.

Absorbance Reading* of Supernatant

Protease Activity

pH of Healthy and OSCCSaliva Samples

pH R

ecor

ded

6.0

6.5

7.0

7.5

8.0

8.5

1. Optimal Variables for Proteolitic Activity

In healthy saliva supernatant, azo label-peptide released by proteases yield a linear dependence to the sample amount. Absorbance reading after two hour incubation

0 2 4 6 8 10 120.000

2.000

4.000

6.000

8.000

10.000

12.000

R² = 0

Supernatant Saliva Vol. (ul)

Abs.

@ 4

50Various Amount Saliva Vs. Average Absorbance Protease Activity in Various pH Buffers

Abso

rban

ce a

t 450

nm

pH 5 pH 6 pH 7 pH 8 pH 90.00

0.05

0.10

0.15

Trypsin

Blank

Saliva supernatant protease stabilized by Tris buffer (pH 7.85) provided the best condition for proteolytic activity.

2. Salivary Protease Comparison of OSSC and Healthy patients.

A. A trend was observed: Saliva pH of OSCC patients was on average more basic, however findings was not statiscally significant (p<0.05)

T-testp= 0.0732

T- Testp= 0.0633

B. Supernatant saliva proteases of OSCC and healthy patients did not give conclusive differences.

Comparison of Protease Activity inControl and Lesion Saliva Samples

Abso

rban

ce a

t 450

nm

Control Lesion0.00

0.05

0.10

0.15

0.20

Trypsin

Blank

B. Healthy saliva lysed epithelial cells released a substantial amount of protease that is comparable to trypsin.

A. Proteases in WS did not have a negative impact during the customary freeze-thaw cycles; proteases were not destroyed/degraded.

0

0.05

0.1

0.15

0.2

0.25

0.3

Abs.

at

450n

m

Blank Trypsin (Lysed cells) Supernatant WS

Preliminary Lysed Cell Protease Activity

3. Protease Properties within Whole Saliva

Saliva Lysed Cells*

Whole Saliva

Supernatant

Spectrophotometer Perkin Elmer-

1420Multilabel Counter

Proteases

Azocasein Azo-label peptides

TCA

T-testp= 0.8572

Nonspecific protease substrate, azocasein, is hydrolyzed by proteases releasing azo label-peptides that can be quantified by the light (450nm) absorbace.

p= 0.001

0 1 2 30

0.02

0.04

0.06

0.08

0.1

0.12

0.14Protease Activity afterVarious Thaw

Cycles

Frozen SalivaFresh Saliva

Number of Thaw Cycles

Abs.

at

450n

m

TCA*Stops Rxn.

2 Hr. Incubatio

n

METHODSWhole Saliva

Supernatant*

Centrifuge*

Buffer*

2 hr. Incubation*

160ul Azocasein*

Cell Pellet٭Sonicate٭

(Lyse cells)160 ul

Azocasein*

2 Hr. Incubation

*10 min. at 3000 crf and 1min. at 16,000 crf; 300 ul supernatant; 60ul buffer pH 8, 2.7 mg/ml; 37oC incubation; 10% trichloroacetic acid (TCA); spectrophotometer 450nm—NaOH added.

200٭ ul cells (concentration 5x106 cells/ml counted by hemocytometer 0.100mm deep) washed with 1x PBS; 3x 40sec. Ultrasonic Processor 50%; this step is being tested.

Continue

Centrifuge

450 nm