Effects of leptin on the viability of human ovarian cancer cells and changes in cytokine expression levels

Background Obesity is associated with increased mortality among ovarian cancer and is a poor prognostic factor. There are significant links between the leptin hormone, a product of the obesity gene, and the development of ovarian cancer. Leptin is a vital hormone-like cytokine secreted from adipose tissue and is mainly involved in the maintenance of energy homeostasis. It regulates several intracellular signaling pathways and also interacts with various hormones and energy regulators. It acts as a growth factor by stimulating cell proliferation and differentiation and in this way contributes to cancer cell development. The aim of the study was to investigate the effects of leptin on human ovarian cancer cells. Methods In this study, the effects of increasing the concentration of leptin were investigated on the cell viability of OVCAR-3 and MDAH-2774 ovarian cancer lines by MTT assay. Moreover, to elucidate the molecular mechanisms of leptin in ovarian cancer cells, changes in the expression levels of 80 cytokines were evaluated after leptin treatment via a human cytokine antibody array. Results Leptin increases the proliferation of both ovarian cancer cell lines. IL-1 level was increased in OVCAR-3 cells and TGF-β level was increased in MDAH-2774 cells after leptin treatment. A decrease in IL-2, MCP-2/CCL8 and MCP-3/CCL7 levels was detected in both ovarian cancer cell lines with leptin administration. An increase in IL-3 and IL-10 expressions, insulin-like growth factor binding proteins (IGFBP) IGFBP-1, IGFBP-2 and IGFBP-3 levels were detected in both ovarian cancer cell lines with leptin administration. In conclusion; leptin has a proliferative effect on human ovarian cancer cell lines and affects different cytokines in different types of ovarian cancer cells.


I. Introduction
New techniques such as cDNA microarrays have neabled us to analyze global gene expression [1][2][3] . However, almost all cell functions are executed by proteins, which cannot be studied simply through DNA and RNA techniques. Experimental analysis clearly shows disparity can exist between the relative expression levels of mRNA and their corresponding proteins 4 . Therefore, analysis of the proteomic profile is critical The conventional approach to analyzing multiple protein expression levels has been to use 2-D SDS-PAGE coupled with mass spectrometry 5,6 . However, these methods are slow, expensive, labor-intensive and require specialized equipment 7 . Thus, effective study of multiple protein expression levels can be complicated, costly and time-consuming. Moreover, these traditional methods of proteomics are not sensitive enough to detect most cytokines (typically at pg/ml concentrations).
Cytokines, broadly defined as secreted cell-cell signaling proteins distinct from classic hormones or neurotransmitters, play important roles in inflammation, innate immunity, apoptosis, angiogenesis, cell growth and differentiation 7 . They are involved in most disease processes, including cancer, obesity and inflammatory and cardiac diseases.
Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool to study cell signaling pathways. Regulation of cellular processes by cytokines is a complex, dynamic process, often involving multiple proteins. Positive and negative feedback loops, pleiotrophic effects and redundant functions, spatial and temporal expression of or synergistic interactions between multiple cytokines, even regulation via release of soluble forms of membrane-bound receptors, all are common mechanisms modulating the effects of cytokine signaling [8][9][10][11][12][13][14] . As such, unraveling the role of individual cytokines in physiologic or pathologic processes generally requires consideration and detection of multiple cytokines rather than of a single cytokine.
1. More Data, Same or Less Sample: Antibody arrays provide high-content screening using about the same sample volume as traditional ELISA.
2. Global View of Cytokine Expression: Antibody array screening improves the chances for discovering key factors, disease mechanisms, or biomarkers related to cytokine signaling.
3. Similar (sometimes better) Sensitivity: As little as 4 pg/ml of MCP-1 can be detected using the C-Series array format. In contrast, our similar MCP-1 ELISA assay has a sensitivity of 40 pg/ml of MCP-1.
4. Increased Detection Range: ELISA assays typically detect a concentration range of 100-to 1000-fold. However, RayBiotech arrays can, for example, detect IL-2 at concentrations of 25 to 250,000 pg/ml, a range of 10,000-fold.

III. Components and Storage
Store kit at < -20°C immediately upon arrival. Kit must used within the 6 month expiration date.
*Each package contains 2 or 4 membranes **For up to 3 months (unless stated otherwise) or until expiration date

IV. Additional Materials Required
Pipettors, pipet tips and other common lab consumables Orbital shaker or oscillating rocker Tissue paper, blotting paper or chromatography paper Adhesive tape or plastic wrap Distilled or de-ionized water A chemiluminescent blot documentation system: CCD Camera X-Ray Film and a suitable film processor Gel documentation system Or other chemiluminescent detection system capable of imaging a western blot If not using fresh samples, freeze samples as soon as possible after collection. Avoid multiple freeze-thaw cycles. If possible, sub-aliquot samples prior to initial storage. Serum-free or low serum containing media (0.2% FBS/FCS) is recommended. If serum containing media is required, testing an uncultured media sample as a negative control is ideal as many types of sera contain cytokines, growth factors and other proteins. It is strongly recommended to add a protease inhibitor cocktail to cell and tissue lysate samples. Avoid using EDTA as an anti-coagulant for collecting plasma if testing MMPs or other metal-binding proteins. Avoid using hemolyzed serum or plasma as this may interfere with protein detection and/or cause a higher than normal background response. Avoid sonication of 1 ml or less as this can quickly heat and denature proteins. Most samples will not need to be concentrated. If concentration is required, a spin column concentrator with a chilled centrifuge is recommended. Always centrifuge the samples hard after thawing (~10,000 RPM for 2-5 minutes) in order to remove any particulates that could interfere with detection. General tips for preparing serum, plasma, cell culture media, urine, and lysate samples can be viewed on the online Resources page of the website.

NOTE: Optimal sample dilutions and amounts will need to be determined by each researcher empirically but the below recommendations may be used as a starting point. Blocking Buffer (ITEM 2) should be used to dilute samples. Normalize by loading equal amounts of protein per sample.
Cell Cultured Media: Neat (no dilution needed) Serum & Plasma: 2-fold to 10-fold dilution Cell and Tissue Lysates: Load 50 to 500 µg of total protein (after a 5-fold to 10fold dilution to minimize the effects of any detergent(s)). Therefore the original lysate concentration should be 1 to 5 mg/ml. Other Bodily Fluids: Neat or 2-fold to 5-fold dilution

C. Handling Membranes
The antibody printed side of each membrane is marked by a dash (-) or number (#) in the upper left corner. Do not allow membranes to dry out during the experiment or they may become fragile and break OR high and/or uneven background may occur. Grasp membranes by the corners or edges only using forceps. DO NOT touch printed antibody spots.

D. Incubations and Washes
Perform ALL incubation and wash steps under gentle rotation or rocking motion (~0.5 to 1 cycle/sec) using an orbital shaker or oscillating rocker to ensure complete and even reagent/sample coverage. Rocking/rotating too vigorously may cause foaming or bubbles to appear on the membrane surface which should be avoided. All washes and incubations should be performed in the Incubation Tray (ITEM 10) provided in the kit. Cover the Incubation Tray with the lid provided during all incubation steps to avoid evaporation and outside debris contamination. Ensure the membranes are completely covered with sufficient sample or reagent volume during each incubation. Avoid forceful pipetting directly onto the membrane; instead, gently pipette samples and reagents into a corner of each well. Aspirate samples and reagents completely after each step by suctioning off excess liquid with a pipette. Tilting the tray so the liquid moves to a corner and then pipetting is an effective method.
Optional overnight incubations may be performed for the following step to increase overall spot signal intensities: Sample Incubation Biotinylated Antibody Cocktail Incubation HRP-Streptavidin Incubation NOTE: Overnight incubations should be performed at 4°C (also with gentle rocking/shaking). Be aware that longer incubations can also increase the background response so complete liquid removal and washing is critical.

VI. Chemiluminescence Detection Tips
Beginning with adding the detection buffers and ending with exposing the membranes should take no more than 10-15 minutes as the chemiluminescent signals may start to fade at this point. Trying multiple exposure times is recommended to obtain optimum results.
A few seconds to a few minutes is the recommended exposure time range, with 30 seconds to 1 minute being suitable for most samples. 7. Wash Buffer I Wash: Pipette 2 ml of 1X Wash Buffer I into each well and incubate for 5 minutes at room temperature. Repeat this 2 more times for a total of 3 washes using fresh buffer and aspirating out the buffer completely each time.

VII. Component Preparation
8. Wash Buffer II Wash: Pipette 2 ml of 1X Wash Buffer II into each well and incubate for 5 minutes at room temperature. Reapeat this 1 more time for a total of 2 washes using fresh buffer and aspirating out the buffer completely each time.

NOTE: The Biotinylated Antibody Cocktail (ITEM 3) must be prepared before use. See Section VII for details
9. Pipette 1 ml of the prepapred Biotinylated Antibody Cocktail into each well and incubate for 1.5 to 2 hours at room temperature OR overnight at 4°C. 10. Aspirate Biotinylated Antibody Cocktail from each well.

E. Second Wash
11. Wash membranes as directed in Steps 7 and 8.

NOTE: The 1,000X HRP-Streptavidin Concentrate (ITEM 4) must be diluted before use. See section VII for detail.
12. Pipette 2 ml of 1X HRP-Streptavidin into each well and incubate for 2 hours at room temperature OR overnight at 4°C.

NOTE:
Do not allow membranes to dry out during detection.
15. Transfer the membranes, printed side up, onto a sheet of chromatography paper, tissue paper, or blotting paper lying on a flat surface (such as a benchtop).
16. Remove any excess wash buffer by blotting the membrane edges with another piece of paper.
17. Transfer and place the membranes, printed side up, onto a plastic sheet (provided) lying on a flat surface.

NOTE: Multiple membranes can be placed next to each other and fit onto a single plastic sheet. Use additional plastics sheets if necessary.
18. Into a single clean tube, pipette equal volumes (1:1) of Detection Buffer C (ITEM 8) and Detection Buffer D (ITEM 9). Mix well with a pipette.

EXAMPLE: 250 µl of Detection Buffer C + 250 µl of Detection Buffer D = 500 µl (enough for 1 membrane)
19. Gently pipette 500 µl of the Detection Buffer mixture onto each membrane and incubate for 2 minutes at room temperature (DO NOT ROCK OR SHAKE). Immediately afterwards, proceed to Step 20.

NOTE: Exposure should ideally start within 5 minutes after finishing
Step 19 and completed within 10-15 minutes as chemiluminescence signals will fade over time. If necessary, the signals can usually be restored by repeating washing, HRP-Streptavidin and Detection Buffers incubations (Steps 11-19).
20. Place another plastic sheet on top of the membranes by starting at one end and gently "rolling" the flexible plastic sheet across the surface to the opposite end to smooth out any air bubbles. The membranes should now be "sandwiched" between two plastic sheets.

NOTE:
Avoid "sliding" the top plastic sheet along the membranes' printed surface. If using X-ray film, do not use a top plastic sheet so that the membranes can be directly exposed to the film.
21. Transfer the sandwiched membranes to the chemiluminescence imaging system such as a CCD camera (recommended) and expose.

NOTE:
Optimal exposure times will vary so performing multiple exposure times is strongly recommended. See Section VI for additional details.

I. Storage
22. To store, without direct pressure, gently sandwich the membranes between 2 plastic sheets (if not already), tape the sheets together or use plastic wrap to secure them, and store at < -20°C for future reference.

Typical Results obtained with RayBio C-series Antibody Arrays
The preceding figures present typical images obtained with RayBio ® C-Series Antibody Arrays. These membranes were probed with conditioned media from two different cell lines. Membranes were exposed with UVP Bioimaging Epichem 3 Darkroom for 1 minute.
Note the strong signals of the Positive Control Spots in the upper left and lower right corners. (See below for further details on the control spots.) The signal intensity for each antigen-specific antibody spot is proportional to the relative concentration of the antigen in that sample. Comparison of signal intensities for individual antigen-specific antibody spots between and among array images can be used to determine relative differences in expression levels of each analyte sampleto-sample or group-to-group.

A. Control Spots
Positive Control Spots (POS) -Controlled amount of biotinylated antibody printed onto the array. Used for normalization and to orientate the arrays. Negative Control Spots (NEG) -Buffer printed (no antibodies) used to measure the baseline responses. Used for determining the level of non-specific binding of the samples. Blank Spots (BLANK) -Nothing is printed here. Used to measure the background response.

B. Data Extraction
Visual comparison of array images may be sufficient to see differences in relative protein expression. However, most researchers will want to perform numerical comparisons of the signal intensities (or more precisely, signal densities), using 2-D densitometry. Gel/Blot documentation systems and other chemiluminescent or phosphorescent detection systems are usually sold as a package with compatible densitometry software.
Any densitometry software should be sufficient to obtain spot signal densities from your scanned images. One such software program, ImageJ, is available for free from the NIH website along with an array plug-in.
We suggest using the following guidelines when extracting densitometry data from our array images: For each array membrane, identify a single exposure that the exhibits a high signal to noise ratio (strong spot signals and low background response). Strong Positive Control Spot signals but not too strong that they are "bleeding" into one another is ideal. The exposure time does not need to be identical for each array, but Positive Control signals on each array image should have similar intensities. Measure the density of each spot using a circle that is roughly the size of one of the largest spots. Be sure to use the same extraction circle dimensions (area, size, and shape) for measuring the signal densities on every array for which you wish to compare the results. For each spot, use the summed signal density across the entire circle (ie, total signal density per unit area). Once the raw numerical densitometry data is extracted, the background must be subtracted and the data normalized to the Positive Control signals to analyze.

C. Data Analysis
Background Subtraction: Select values which you believe best represent the background. If the background is fairly even throughout the membrane, the Negative Control Spots (NEG) and/or Blank Spots (BLANK) should be similar and are accurate for this purpose.
Positive Control Normalization: The amount of biotinylated antibody printed for each Positive Control Spot is consistent from array to array. As such, the intensity of these Positive Control signals can be used to normalize signal responses for comparison of results across multiple arrays, much like housekeeping genes and proteins are used to normalize results of PCR gels and Western Blots, respectively.
To normalize array data, one array is defined as "Reference Array" to which the other arrays are normalized to. The choice of the Reference Array is arbitrary.

NOTE:
The RayBio ® Analysis Software Tools always designate Array 1/Sample 1 as the Reference Array.
Next, the simple algorithm below can be used to calculate and determine the signal fold expression between like analytes.

X(Ny) = X(y) * P1/P(y)
Where: P1=mean signal density of Positive Control spots on reference array P(y)=mean signal density of Positive Control spots on Array "y" X(y)= X(Ny)=normalized signal intensity for spot "X" on Array "y" For example: Let's determine the relative expression of IL-6 on two different arrays (Arrays 1 and 2). Let's assume that the duplicate signals for the IL-6 spots on each array are identical (or that the signal intensity used in the following calculation is the mean of the two duplicates spots). Also assume the following: P1 = 2500 P2 = 2700 IL-6 (1) = 300 IL-6 (2) = 455 Then IL-6(N2) = 455 *2500/2700 = 421.30 The fold increase of IL-6(N2) vs IL-6(1) = 421.3/300 = 1.40-fold increase or a 40% increase in the signal intensity of IL-6 in Array 2 vs. Array 1. Ensure all the wash steps are carried out and the wash buffer is removed completely after each wash step Non-specific binding Ensure the blocking buffer is stored and used properly