Curcumin and Vitamin E Protect against Adverse Effects of Benzo[a]pyrene in Lung Epithelial - PowerPoint Presentation

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Curcumin and Vitamin E Protect against Adverse Effects of Benzo[a]pyrene in Lung Epithelial Cells

Wenbin

z,

meghan

mc,

qingsong

c,

tangfeng

l,

kamaleshwar

s,

weimin

g

Danielle Irby

Doctor of pharmacy candidate 2019

Mentor: Dr. Yogesh Kulkarni

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Background Information

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Lung Cancer

Lung cancer is the deadliest cancer among both men and women. About 1 out of 4 cancer-related deaths are from lung cancer. The American Cancer Society projects there to be 222,500 new cases and 155,870 deaths from lung cancer in 2017.

Etiology

Tobacco smoking

Including secondhand smoke

Environmental and workplace exposure

Treatment

varies based on the stage of cancer the patient is in. Options can range surgery to chemotherapy and radiation therapy.

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Benzo[a]pyrene

Benzo[a]pyrene (

BaP

) is a polycyclic aromatic hydrocarbon that acts as one of the major carcinogens in cigarette smoke.

IARC 1

Carcinogen

Metabolized in to Ba-P-7,8-diol-9,10-epoxide (BPDE)

BPDE, the carcinogenic form of

BaP

, can either be metabolized or form adducts with DNA and cause DNA damage

Research reveals

BaP

also induces oxidative stress

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Curcumin

Active compound found in Turmeric

Turmeric (

Curcuma longa

) has long been used for medicinal purposes, such as fighting infection, inflammation reduction and digestive problems. It is cooked in many foods, and known for giving Indian curry its flavor and yellow color.

Powerful antioxidant – scavenger of superoxide O2- and hydrogen peroxide H2O2

Studies have shown curcumin to have

chemopreventive

effects

fat-soluble antioxidant

α

-tocopherol, used in this study, is one of abundant forms of vitamin E and is the most biologically active variant

Vitamin E

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Materials

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Materials

Curcumin (98% pure),

BaP

, α-tocopherol succinate, and DMSO (Sigma Chemical Co.: MO, USA). Stock concentrations of 50 µM for curcumin and VE.

BaP-tetrol

standards were purchased from

MRIGlobal

Chemical Carcinogen Reference Standard Repository (MO, USA)

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) purchased from

Usb Corporation. (CA, USA)DNEasy Blood and Tissue Kits (QIAGEN: MD, USA)

Acetonitrile (ACN) and ammonium

formate

(AF) were purchased from Merck (Darmstadt, Germany).

HCl was from Fischer Scientific (Fair Lawn, NJ)

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Cell Culture

BEAS-2B

Human bronchial epithelial cell line

American Type Culture Collection (ATCC)

Cultured in LHC-9 (Life Technology, MI) complete growth medium supplemented with penicillin and streptomycin.

Incubate at 37°C in 5% CO

2

.

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Purpose

…“to elucidate the role of curcumin and VE (vitamin E) in cellular remediation after acute exposure to

BaP.

”

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Methods and Results

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Methods: MTT Assay

MTT assays measure cell viability through the quantification of formazan production in a sample of cells. Viable cells can reduce MTT to formazan, which is purple in color.

cells/well

Treatments:

BaP

alone (0-80 µM) for 24 and 48h

Curcumin (0-80 µM) or VE individually for 24, 48, and 72h

Co-treatment with

BaP

(5

µM) and VE (0-80 µM) for 24h

Co-treatment with

BaP

(5 µM) and curcumin (0-80 µM) for 24h

MTT solution (5mg/mL) was added and incubated with the cells for 3h.

After solubilizing with DMSO, absorbance was read at 570 nm.

 

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Drug Treatments

Using results from the MTT assay further treatments were done using:

5 µM

BaP

5 µM Curcumin

20 µM VE

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ROS Assay

Reactive oxygen species formation was detected using 2,7-dichlorofluorescein diacetate (DCFDA) (Sigma Chemical Co., MO). This non-fluorescent probe is de-

esterfied

intracellularly to form 2,7-dichlorofluorescein.

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ROS Assay continued

96-well plate

Treated with previously mentioned drug treatments for 24h

Washed with 200 µL PBS

Incubated with 100 µL of 20 µM DCFDA solution for 45 min at 37°C

Measured fluorescence released: excitation 485 nm, emission 535 nm

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Flow Cytometry

Flow cytometry was used to measure the percentage of cells in the pre-G1, G0/G1, S and G2/M phases of the cell

cyle

.

cells from each treatment group collected

Fixed in 70% ethanol for >24h at 4°C

Stained with Guava Cell Cycle Reagent and run on a Guava

EasyCyte

Flow Cytometer (Millipore Corporation, MA)

events were counted and phase percentages were determined using

GuavaSoft

software (Millipore)

Each sample was run in triplicate

 

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Flow Cytometry continued

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DNA adduct analysis

Adduct presence was measured using high-performance liquid chromatography (HPLC)

of 24h treated cells

Trypsinized

and,

DNEasy

Blood and Tissue kit, were used to extract DNA

DNA quantity estimated using Nano-Drop 1000

Spectrophotmeter

at 260/280 nm

Samples were dissolved in 0.1 N HCL and acid hydrolyzed at 90°C for 3h

BPDE

tetrol

adducts were determined by HPLC

60 µL per sample, injected onto a C18 column

Eluted for 32 min

1 mL/min

Mobile phase: 90:10 (A:B), Solvent A (5% ACN with 10

mM

AF), Solvent B (100% ACN with 10

mM

AF)

Excitation 344 nm, emission 398 nm

 

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DNA adduct analysis cont.

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qRT

-PCR

Used one-step real time PCR kit with SYBR green for amplification of total RNA (50 ng)

Single-step amplifications performed using

iCycler

IQ (Bio-Rad)*

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Western blot analysis

Technique used to detect and quantify specific proteins in a sample of cells or tissue homogenates. In this study, immunoblotting was performed using phospho-p53 (Ser 15) (p-p53), p53, PARP-1, COMT, SOD-1 and

survivin

.

24h treatment, washed and lysed with RIPA lysis buffer

BCA Assay using Bradford protein assay (Bio-Rad)

12% SDS polyacrylamide gel electrophoresis

Transferred to polyvinylidene fluoride (PVDF) membranes

Blocked with 3% milk, then probed with primary antibody (Santa Cruz Biotechnology) overnight at 4°C (α-tubulin was used as an internal control)

Washed and incubated with secondary antibody (Cell Signaling) for 1h at room temperature

Briefly incubated with chemiluminescence then exposed to X-ray film (Fujifilm Corp., Tokyo)

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Statistical Analysis of Results

Analysis of variance (ANOVA) for cell viability tests, ROS production and DNA adducts.

Probit

analysis was performed to calculate IC50s.

One-way ANOVA was used to determine the difference in gene and protein expression between groups

Differences at P<0.05 were considered statistically significant

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Discussion and Conclusions

As displayed by the results, 5 µM

BaP

is cytotoxic and can induce DNA damage even at acute exposure (24h)

Curcumin has pleiotropic effects on cell death and survival, while Vitamin E exhibits biphasic qualities

Antioxidants, curcumin and VE, are

cytoprotective

against

BaP

-induced cytotoxicity in BEAS-2B cells by

attenuating ROS production

Reducing the

BaP

-induced increase in activated p53 activation and PARP-1*Reversal of BaP-induced survivin down-regulation (Curcumin)Significant reduction of BPDE-DNA adduct formation

The first study to report the protective effects of VE against DNA damage caused by short-term

BaP

exposure

Curcumin and VE do not reduce

BaP

-induced DNA damage through the regulation of CYP genes involved in

BaP

metabolism

*proposed to be attributed to the reduced oxidative stress and DNA damage in

BaP

-treated cells by Curcumin and VE treatment

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References

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092992

https://www.ncbi.nlm.nih.gov/books/NBK22268/

https://pubchem.ncbi.nlm.nih.gov/compound/benzo_a_pyrene#section=Wikipedia

http://www.umm.edu/health/medical/altmed/herb/turmeric

http://www.sigmaaldrich.com/catalog/product/sigma/d6883?lang=en&region=US

https://www.bio.umass.edu/micro/immunology/facs542/facswhat.htm

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Q&A