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Hypothesis Inhibi.on of HMOX-‐1 through ShRNA will lead to an increased effec.veness of gemcitabine and thereby augment cancer cell death. Similarly, overexpression of HMOX-‐1 through the same method will lead to a decrease in effec.veness and lower death count of cancer cells.
Experimental Method
PI staining gives the advantage of dis.nguishing between living and dead cells, as DNA of dead cells will be fluorescent and living ones will not. Also, it allows us to use flow cytometry to analyze the cell count based on their physical and chemical characteris.cs.
Introduc.on One in nine Canadian women is expected to develop breast cancer in her life.me (1). Heme-‐oxygenase-‐1 (HMOX-‐1) is an enzyme that degrades pro-‐oxidant heme, an iron containing compound present in hemoglobin, and other hemoproteins. The products of this reac.on have an.oxidant proper.es, which protect cell membranes from reac.ve oxygen species. This effect can be exploited by cancerous cells into protec.ng themselves against chemotherapy drugs such as gemcitabine, which cause oxida.ve stress to cells.
Ra.onale
An.cipated Results Assuming HMOX-‐1 decreases the effec.veness of gemcitabine treatment in 4T1 mouse breast cancer cells, the sample with HMOX-‐1 knockdown would have the highest count of fluorescent (dead) cells present, followed by the controlled sample. Lastly, the sample with HMOX-‐1 overexpression would give the lowest count of fluorescent cells, indica.ng reduced efficiency of gemcitabine in trea.ng breast cancer.
Discussion The experiment will be carried in vitro, and past experiments have shown that HMOX-‐1 may behave differently in vivo than it does in vitro (4,7). This suggests that further experiments should be carried out researching the difference in vivo. If the results of this experiment are conclusive, then the informa.on could be applicable to other chemotherapy agents or other types of cancer. In addi.on, HMOX-‐1 chemical inhibitors, such as zinc or .n protoporphyrin (ZnPPIX, or SnPPIX), could be used along with gemcitabine or other chemotherapy agents to work synergis.cally and produce growth inhibi.on in gemcitabine-‐resistant breast cancer cells.
References (1) Canadian breast cancer founda.on. (2013). Retrieved from hYps://www.cbcf.org/central/Pages/default.aspx. (2) Lau, Alexandria, Nicole V., Zheng S., Pak K., and Donna D. "Dual Roles of Nrf2 in Cancer." Pharmacological Research 58.5-‐6
(2008): 262-‐70 (3) Nuhn, P., Kunzli, B., & Hennig, R.,et al (2009). Heme oxygenase-‐1 and its metabolites affect pancrea.c tumor growth in vivo. Mol Cancer, 8(7),
37-‐43. (4)Was, H., Jozef, D., & Alicja, J. (2012). Heme oxygenase-‐1 in tumor biology and therapy. Current Drug Targets, 11(12),
1551-‐1570. (5) Nowis, D., et.al06). Heme oxygenase-‐1 protects tumor cells against photodynamic therapy-‐mediated cytotoxicity. Oncogene,
25(24), 3365-‐3374. (6) Berberat, P., et.al (2005). Inhibi.on of heme oxygenase-‐1 increases responsiveness of pancrea.c cancer cells to an.cancer
treatment. Clinical Cancer Research, 10(11), 3790. (7) Ryter, S., Alam, J., & Choi, A. (2006). Heme oxygenase-‐1/carbon monoxide: From basic science to therapeu.c applica.ons.
Physiological Reviews, 86(85), 583-‐650. (8) Song W, Su H, Song S, Paudel HK, Schipper HM. Over-‐expression of heme oxygenase-‐1 promotes oxida.ve mitochondrial
damage in rat astroglia. J Cell Physiol 2006; 206: 655-‐63. Image (a)DNA photo: hYp://watchdog.wpengine.netdna-‐cdn.com/wp-‐content/blogs.dir/1/files/2013/11/shuYerstock_61775431.jpg Image (b) Petri dish photo: hYp://www.clker.com/clipart-‐342081.html Figure 1 Kim, Ada. Unpublished data.
HMOX-‐1 knocked down
Murine mammary 4T1 carcinoma cells
Inhibi.on by shRNA Overexpression by an expression vector
HMOX-‐1 overexpressed Control
Chemotherapy agents such as gemcitabine are stress factors for cancer
cells.
HMOX-‐1 is a key enzyme for protec.on against oxida.ve stress, providing resistance to chemotherapy (6,7)
Inhibi.ng HMOX-‐1 will lower the defense of cancer cells, making gemcitabine more effec.ve (8)
Inhibited Control Overexpressed
Arbitrary Num
ber
Living Cells A4er Gemcitabine Treatment
Acknowledgement We would like to thank Ada Kim-‐ our mentor, and the URO team for all the help they have provided.
Figure 1. Hypothesized Results: Higher number of living cancer cells amer treatment with gemcitabine in the
overexpressed group, and lowest in the inhibited group
Treat the cell cultures with gemcitabine
Analyze viability using Propidium Iodide (PI) staining and flow
cytometry
Analyze cell cultures for presence or absence of HMOX-‐1 using Western
Blonng
Image (a)
Image (b)
heme + NAD(P)H + H+ + 3 O2 ↔ biliverdin + Fe2+ + CO + NAD(P)+ + 3 H2O (2)
Figure 1b: Western Blot showing inhibi.on of HMOX-‐1 in 4T1 murine mammary carcinoma cells
Figure 1a: Western Blot showing overexpression of HMOX-‐1 in 4T1 murine mammary carcinoma cells
Previous Research This rela.onship has been studied with regards to some types of cancer: high HMOX1 expression in pancrea.c cancer cell lines was associated with increased chemoresistance to gemcitabine (3), but other studies have shown that inhibi.on of HMOX-‐1 does not necessarily lead to increased effec.veness of treatment in all types of cancer(4,5). However, the cytoprotec.ve role of HMOX1 amer chemotherapeu.c treatment is unknown in breast cancer.
