1. NSAIDs therapy for colorectal cancer: mechanisms and efficacy
PHM Fall 2016
Coordinator: Dr. Jeffrey Henderson
Instructor: Dr. David Hampson
NSAIDs therapy for colorectal cancer: mechanisms and efficacy
PHM142
Sign up date: Oct 17, 2017
Ji Hyun (Sera) Lee
Cathy Hoang
Janet Chan
Rui Si (Louise) Pi

2. What are NSAIDs? → Non-steroidal anti-inflammatory drug
→ Commonly used for fever, inflammation, pain
→ Examples: Acetylsalicylic Acid (Aspirin), Ibuprofen (Advil), Naproxen (Aleve)
→ Some available as OTC

3. NSAIDs’ Molecular Targets - COX enzyme
→ Cyclooxygenase enzyme (COX) responsible for the production of prostaglandins via arachidonic acid
→ Prostaglandins will activate the inflammatory response, resulting in fever/pain
→ Also play a role in constricting blood vessels, aggregating platelets (blood clotting)
→ NSAIDs bind to active sites on COX, thereby preventing the formation of prostaglandins, and reducing inflammation, fever, and pain
→ Aspirin irreversibly acetylates COX binding site, resulting in blood thinning, and less clotting

4. → Prostaglandin synthesis pathway
→ Production of various prostaglandins and thromboxanes
→ Thromboxane aids in blood clotting and vasoconstriction of vessels

5. Colorectal Cancer and Wnt-Signaling pathway
→ 2nd most commonly diagnosed cancer in Canada
→ On average, 26 Canadians die from colorectal cancer daily
→ Malignant tumour starts in colon/rectum
→ NSAIDs can aid in reducing cell proliferation and thus preventing cancer
under normal physiology, wnt signaling protein would bind to the g-protein, no complex is made of APC-axin-GSK, resulting in beta-catenin not degraded so it can travel into the nucleus and turn on transcripton for cell proliferation.
when theres no wnt, beta-catenin is targeted for degradation by APC-axin-GSK complex→ B catenin is phosphorylated + degraded = no cell proliferation
in cancer pts, mutation on APC gene --> APC loss of function = no complete complex + no phosphorylation of B catenin --> so beta-catenin will never get degraded --> uncontrolled cell proliferation

6. Inhibition of COX2: immune response suppression and subsequent inhibition of Wnt-signalling pathway
PGE2 promotes malignant cell growth by mediating immune response
Mucosal injury on the colorectal epithelium of CRC patient
Immune cells being recruited to site of injury
Cytokines (IL-alpha, IL-beta, interferon-gamma) released
Activation of COX2 and PGE2 synthesis
PGE2 binds to its receptor EP2 (G-protein coupled receptor)
Complexing of axin, APC and G-protein
Accumulation of beta-catenin in the cytosol and nucleus
Beta-catenin activates the transcription of its target genes, leading to uncontrolled cancerous cell proliferation
EP2
As we mentioned, NSAIDs could inactivate COX-2, thereby inhibiting prostaglandin E2 synthesis (PGE2)
As sera mentioned, in colorectal cancer pts, the Wnt-signalling pathway is unregulated, leading to uncontrolled proliferation of cancerous cells
It was found that PGE2 plays an important role in promoting cancerous cell growth by mediating an immune response and by strengthening the Wnt-signalling pathway
(left) when there is mucosal injury in the colorectal epithelium, immune cells(such as neutrophils, would be recruited to the site of injury
These immune cells then release cytokines (such as interleukin 1 IL‑1α and β, interferon‑γ and tumour necrosis factor (TNF))
In response to those cytokines, COX2 will then be activated and produce PGE2
PGE2 would bind to its receptor EP2 (a G-protein coupled receptor), the binding would lead to the complexing of Axin, APC and the g protein (G alpha S)
This will result in the buildup of beta-catenin in the cytosol and nucleus
Once in the nucleus → uncontrolled cellular proliferation, and angiogenesis

7. Inhibition of COX2: immune response suppression and subsequent inhibition of Wnt-signalling pathway
Under NSAID therapy
COX2 inhibition → PGE2 synthesis ↓
Unbound EP2 liberates APC and axin
APC-axin-GSK complex phosphorylates beta-catenin, following by its degradation
Without beta-catenin, the tumorigenesis transcription machinery is turned off
EP2
In the presence of NSAID, COX2 would be inhibited thereby reducing the production of PGE2
When PGE2 is not bound to the G-protein coupled receptor, Axin and APC are liberated, and they can form a complex with another protein GSK-3beta
This new complex would phosphorylate beta-catenin, followed by the degradation of beta-catenin
w/o beta-catenin, the tumorigenesis transcription machinery will be turned off

8. Inhibition of COX1: Platelet inhibition and PEG2 synthesis suppression
Platelets
Host immune cells
Express COX1 only
Not nucleated; cannot resynthesize COX1 when inhibited
Convert AA to thromboxane TXA2 by COX1
TXA2
Stimulates activation of new platelets as well as increases platelet aggregation
Results in more platelets at site of injury and increases PGE2 level
With NSAID therapy
COX1 inhibition → TXA2 synthesis ↓ → less platelet aggregation at site of injury → COX2 activation and PGE2 synthesis ↓ → Wnt-signalling pathway inactivated → tumorigenesis suppression
NSAID
COX1
Platelet inhibition
Unlike epithelial cells which express both COX-1 and 2
Platelet only expresses COX1
And because it’s not nucleated, it cannot resynthesize COX1 when it is inhibited
COX1 converts AA to thromboxane TXA2 (TXA2 stimulates activation of new platelets as well as increases platelet aggregation) - like a positive feedback mechanism
Platelets (one of the host immune cells that could activate COX-2 and subsequent PGE2 production)
When NSAID is present, inhibits COX-1 → reduce synthesis of TXA2 → less platelet aggregation + no activation of new platelets → no COX-2 activation and reduced PGE2 production → no Wnt-signalling pathway not activated → no cell proliferation

9. Efficacy of NSAIDs on Colorectal cancer
Study by Sheng et al.,
Growth of human colon cancer cell (HCA-7) that express COX2 in nude mice +/- treatment with selective COX2 inhibitor (SC-58125)
COX2 enzyme converts arachidonic acid to prostaglandin: prostaglandin production was significant in HCA-7, but was inhibited by COX2 inhibitor treatment
With the addition of COX2 inhibitor, it inhibited the size and number of colonies derived from COX2 expressing HCA-7
Treatment of COX2 inhibitor inhibited tumor development by 90% in HCA-7
This particular study showed how effective NSAIDs is on the treatment of colorectal cancer. The experiment was conducted using growth of human colon cancer cells that constitutively express COX-2 enzyme in nude mice with and without treatment of a highly selective COX-2 inhibitor.
Results showed that prostaglandin production was significant in colon cancer cells and this was inhibited by COX-2 inhibitor treatment.
Next, non-transformed intestinal epithelial cells are unable to survive when plated in extracellular matrix component. On the other hand, colon cancer cells are able to form discrete colonies when plated. With the addition of COX-2 inhibitor, it dramatically inhibited the size and number of colonies derived from COX-2 expressing colon cancer cells.
Finally, treatment of COX-2 inhibitor inhibited tumor development by 90% for human colon cancer cell in the mice. Therefore, COX-2 inhibitor can inhibit colon cancer cell growth in culture as well as tumor implanted in nude mice.

10. Efficacy of NSAIDs on Colorectal cancer
Epidemiologic studies show a 40-50% reduction in mortality from colorectal cancer in individuals taking NSAIDs, compared to those not using it
Patients with familial adenomatous polyposis (FAP) who take sulindac have a significant reduction in adenoma size and number
Epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in individuals taking NSAIDs, such as aspirin, compared to those not taking these agents
And patients with familial adenomatous polyposis (FAP) who take sulindac, also a NSAIDs medication, have a significant reduction in adenoma size and number.

11. Adverse Effects of NSAIDS
→ Varies depending on the inhibited COX enzyme(s)
→ GI Mucosal Effects (COX1 inhibition)
→ Renal Effects (COX1&2 inhibition)
→ Cardiovascular Effects (COX1&2 inhibition)
There are a lot of adverse effects or side effects that come with using NSAIDs
Three large categories of adverse effects are concerning the GI mucosa, kidney and cardiovascular system
The effect depends on the COX enzyme that is inhibited

12. COX1 inhibition = GI bleeding and peptic ulcers
GI Mucosal Effects
→ COX1 is involved with gastric protection
→ COX1 produces PGE2
→ Increases mucus secretion
→ Increases bicarbonate secretion
→ Increases mucosal blood flow
→ Inhibition of COX1/PGE2 production = mucosal and epithelial damage
COX1 inhibition = GI bleeding and peptic ulcers
COX1 is involved with gastric protection by producing prostagladin E2
Prostaglandin E2 is involved with increasing mucus secretion, bicarbonate secretion and mucosal blood flow
By using NSAIDs, COX1 will be inhibited, which subsequently inhibits prostaglandin E2
This leads to muscoal and epithelial damage, and ultimately GI bleeding and peptic ulcers
This resulted in the development of selective inhibitors of COX2, such as celecoxib, which has been shown to be effective for colorectal cancer

13. COX1&2 inhibition = Hypertension and hemodynamic acute kidney injury
Renal Effects
→ COX1&2 involved in PGE2 and PGI2 production
→ PGE2 and PGI2 have effects on kidneys
→ Increased sodium and water excretion
→ Increased afferent arteriolar vasodilation
→ Inhibition of COX1&2 = sodium and water retention and decreased GFR
COX1&2 inhibition = Hypertension and hemodynamic acute kidney injury
In terms of renal effects, both COX enzymes are involved
COX1 and 2 are responsible for producing prostaglandin E2 and I2
These prostaglandins increase sodium and water excretion, as well as vasodilation of the afferent arteriole
Inhibition of COX1 and 2 therefore lead to sodium and water retention and a decrease in glomerular filtration rate
Thus, the result of using NSAIDs is hypertension and hemodynamic acute kidney injury since renal perfusion is not maintained

14. Cardiovascular Effects
→ COX1 involved in TXA2 production and COX2 involved in PGI2 production
→ TXA2 effects
→ Increased platelet aggregation
→ Increased vasoconstriction
→ PGI2 effects
→ Decreased platelet aggregation
→ Increased vasodilation
Inhibition of COX2 = disrupted balance = cardiovascular risks
Finally, both COX enzymes are also involved cardiovascular effects
COX1 produces thromboxane A2 and COX2 produces prostaglandin I2
Thromboxane increases platelet aggregation and vasoconstriction, whereas prostaglandin I2 decreases platelet aggregation and increases vasodilation
By inhibiting COX2 with an NSAID, the balance between thromboxane A2 and prostaglandin I2 is disrupted

15. Cardiovascular Effects
Thromboxane A2 accumulates
Clumping will occur and blood vessels with be constricted
This can lead to CV risks, such as myocardial infarctions
People were focused on preventing GI effects, so they created selective COX2 inhibitors, but didn't look into cardiovascular effects
But of course it is important to take this cardiovascular effect into account

16. Summary
NSAIDs bind to COX resulting in reduced inflammation, pain, fever, thinned blood, and blood clotting
In colorectal cancer patients, APC is absent, thereby preventing the degradation of beta-catenin which results in cell proliferation
PGE2 synthesis induced by inflammation would activate Wnt-signaling pathway, resulting in tumorigenesis
Inhibition of COX2 by NSAIDs leads to reduced PGE2 synthesis, beta-catenin degradation and reduced tumorigenesis
Inhibition of COX1 by NSAIDs reduces TXA2 synthesis and further platelets activation, COX2 is not activated to produce PGE2, thereby inhibiting Wnt-signalling pathway and malignant cell proliferation
COX-2 inhibitor decreases prostaglandin production in colon cancer cells, and inhibits tumor development
There is a 40-50% reduction in mortality from colorectal cancer in individuals taking NSAIDs
Inhibition of COX by enzymes has GI mucosal, renal and cardiovascular effects
Inhibition of COX1 can lead to GI bleeding and peptic ulcers
Inhibition of COX1 and 2 can result in hypertension and hemodynamic acute kidney injury
Inhibition of COX1 and 2 can cause myocardial infarctions

17. References
Sheng, H., Shao, J., Kirkland, S. C., Isakson, P., Coffey, R. J., Morrow, J., … DuBois, R. N. (1997). Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2. J Clin Invest, 99(9), 2254–9.
DuBois, R. N., & Smalley, W. E. (1996). Cyclooxygenase, NSAIDs, and colorectal cancer. Journal of Gastroenterology.
Canadian Cancer Society's Advisory Committee on Cancer Statistics. (2017). Canadian Cancer Statistics Toronto, ON: Canadian Cancer Society
Ricciotti, E., & Fitzgerald, G. A. (2011). Prostaglandins and inflammation. Arteriosclerosis, Thrombosis, and Vascular Biology, 31(5), 986–
Abdulghafar, A. Mechanism of action of Aspirin as antiplatelet and its different doses. Retrieved from:
NSAIDS (nonsteroidal anti-inflammatory drugs). Retrieved from:
Major Side Effects of NSAIDs & COX-2 Selective Inhibitors (2017, March 10). Retrieved from:
Clevers, H. (2006). Colon Cancer — Understanding How NSAIDs Work. New England Journal of Medicine, 354(7), 761–763.
Drew, D. A., Cao, Y., & Chan, A. T. (2016). Aspirin and colorectal cancer: the promise of precision chemoprevention. Nature Reviews Cancer, 16(3), 173–186.
Weinberg, J., Fermor, B., & Guilak, F. (2007). Nitric oxide synthase and cyclooxygenase interactions in cartilage and meniscus: relationships to joint physiology, arthritis, and tissue repair. Sub-Cellular Biochemistry, 42, 31–62.