Evaluation of Research Article

8/12/2019 Evaluation of Research Article

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Cancer Biology

Evaluation of Research Article

NOSH-aspirin (NBS-1120), a novel nitric-oxide- and hydrogensulfide-releasing hybrid is a potent inhibitor of colon cancer cellgrowth in vitro and in a xenograft mouse model by Kashfi et al.

Done by:Nishachand Vohreh (09034669)

BBSD1 10121A


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Cancertherapeutic

agent againstcolon cancer

Higher dosagehas lead to

gastrointestinalproblem

Damage to theepithelial of thegastric mucosa

Nitric oxide (NO)and hydrogensulfide (H2S)

reduce gastrictoxicity

NO is anti-inflammatory

H2SH2S ability ofthis tissue toresist damage

Hybrid of Aspirin(NOSH) have

been synthesized

Aspirin


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Synthesized fourclasses of NOSH-

ASA

Investigated theiranti-cancer and anti-

inflammatory

properties

NOSH-1 was morepotent

Specifically using

NOSH-ASA 1 againstonly colon cancer cells

lines (HT-29)

Previous works on hybrid?


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Methods

Methylene Bluemethod to

measure H2S

Effect oftumour size

using male nudemice

Calorimetric COXinhibitor screening

kit to measureeffect on COX 1

and 2

Flow cytometry(PI for Cell

cycle) (AnnexinV for apoptosis)

Nitrate/Nitritecalorimetric

assay measureamount of NO

Elisa tomeasure cellproliferation

MTT assay tomeasure cell

viability


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Cell Growth Inhibition Compared the effects of Aspirin(ASA) and the

hybrid NOSH-ASA

Measured the viability of cells after being

incubated with the drugs

At a lower dose, NOSH-ASA was able to stop50% of the cells in culture to stop growing as

compared to ASA (IC50:0.05um)


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Cell Growth Inhibition


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Cell Growth Inhibition

NOSH-ASA was said to be 250,000 times morepotent than ASA at 72 hours

Compared the potency of NOSH-ASA with HS-ASA and NO-ASA

The new hybrid proved to be more potent, 80 and15000 times more potent respectively

Here, they realise the importance of (H2S) It had the closest IC50:4um


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Cell Growth InhibitionTreatment IC50 uM Fold-enhanced

potency of

NOSHaspirin

ASA >5000 >100,000

p-NOASA 10 2 ~200

o-NOASA 8 3 ~160

m-NOASA 185 15 ~3700

NOASA (aliphatic

spacer)

750 35 ~15,000

HSASA 4 0.5* ~80

NOSHaspirin 0.05 0.003 -

Comparison in IC50 values for cell growth inhibition between aspirin,

NOaspirin, HSaspirin, and NOSHaspirin


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Cancer Cell KineticsProliferation

Evaluated the effects of NOSH-ASA on cellproliferation, transition in the cell cycle andapoptosis

Using Proliferating Cell Nuclear Antigen as aplatform for Bromodeoxyuridin (BrdU), % ofproliferating cells were measured


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Proliferation


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Cell Cycle Phase Using flow cytometry and Propidium Iodidestaining of the nucleus, obtained percentage of

cells in the different stages of the cell cycle

More cells have to stop replicating (S phase)and remain in the G1 phase


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Cell Cycle Phase


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Apoptosis

Drug should be able to induce death in thecancer cells

Flow cytometry but with Annexin V staining

of phosphatidylserine obtained percentagesof cells going through apoptosis


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Apoptosis


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Does NOSH-ASA really liberate H2Sand NO?


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Actual rate of H2S released?

NOSH-ASA and HS-ASA were used

One set was used in the culture medium ofcell lines while the other was incubated in rat

liver

The rate of release for both was higher inthe rat liver when compared to the culturemedia

However, comparing between NOSH-ASA andHS-ASA, HS-ASA released H2S at a higherrate in the liver


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Actual rate of H2S released? This might be due to many enzyme activities

going on in the liver

Slower rate of release of H2S from NOSH-

ASA would mean more Hydrogen sulphideexposed to cells

Potency of NOSH-ASA increase


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Is the value of ASA still present in theHybrid?

Aspirin used as a pain killer by inhibitingCyclooxygenase (COX )

COX-1 and COX-2 were used to test theability of Aspirin as ASA and NOSH-ASA

Indomethacin as a control


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Is the value of ASA still present in theHybrid?

COX was inhibited in both but a smaller amountof NOSH-ASA was needed to inhibit more ofCOX

NOSH-ASA inhibits more of COX-1 ascompared to ASA

As a hybrid, aspirins value increase whichmight be due to the synergy effects of the

other molecules

As such, this form of ASA could be used asa commercial pain killer but at lower dose


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Same effects if H2S and NO areseparated from NOSH-ASA and

tested? Evaluate the effects of the intact NOSH-

ASA molecules and parts

A combination of ASA and H2S(ADT-OH)/NO(SNAP) and compared growthinhibition

Most noticeable was the difference betweenNOSH-ASA and ASA + SNAP + ADTOH


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IC50values for cell growth inhibition by various components ofNOSHaspirin or other agents that release NO or H2S alone or

in combination

Same effects if H2S and NO areseparated from NOSH-ASA and

tested?

Compound IC50 at 24 h, uM

ASA >1000

SNAP 530 45

ADTOH 26 3

ASA + SNAP 710 35

ASA + ADTOH 380 45

ASA + SNAP + ADTOH 450 35

NOSHASA 0.05 0.005


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8 immuno-comprimised mice were used

Heterotrophic models by injecting the cells

subcutaneously

4 control and 4 were treated

Treated mice 80% reduction in tumour sizewith no adverse side effects. Weight of mice remained constant throughout

Heterotrophic Xenograft studies


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Heterotrophic Xenograft studies


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Future of Drug

At the end of this experiment, theresearchers realise that this new hybrid hasgood potential in the treatment of coloncancer

A small dose as found, might reduce the riskof unwanted side effects like seen in thetraditional aspirin

No other NSAID have been found to havesuch a high degree of potency


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Future of Drug

The search to make aspirin a safer drug has ledto the formation of a potent anti-cancer agent

Experiments on other cell lines might be the

next step for the researchers

This drug is still in the development phase andneeds more trials to make it available forhuman use


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References

Chattopadhyay, M., Kashfi, K., and Kodela, R. (2012). NOSH-Aspirin: A Novel Nitric OxideHydrogen Sulfide-ReleasingHybrid: A New Class of Anti-inflammatory Pharmaceuticals.ACS Medical Chemistry Letters 3, 257-262.

Chattopadhyay, M., Kashfi, K., Kodela, R. and Olson, K. (2012).NOSHaspirin (NBS-1120), a novel nitric oxide- and hydrogen

sulfide-releasing hybrid is a potent inhibitor of colon cancercell growth in vitro and in a xenograft mouse model.Department Biochemical and Biophysical ResearchCommunications 419, 523-528.

Heath, W., Namboodiri, M.M. and Thun, M.J. (1991). Aspirinuse and reduced risk of fatal colon cancer. The New EnglandJournal of Medicine 324(23), 1593-1596.

DuBois, R.N., Hori, M., Kawano, S., Sawaoka, H., Tsuji, M., andTsuji, S. (1998). Cyclooxygenase Regulates AngiogenesisInduced by Colon Cancer Cells. Cell93, 705-716.

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