It was demonstrated previously in NIH3T3 cells that cells lost focal adhesions upon treatment with cordycepin at 50µM (Wong et al., 2010). As we had already seen effects of cordycepin on polyadenylation and cell proliferation we next examined the effects cordycepin had on cell shape. In order to study changes in cell shape in detail, immunohistochemistry was performed using the focal adhesion marker vinculin. MCF-7 cells were seeded on cover slips and were treated after 24 hours with cordycepin 50µM for 3 hours. As observed in figure 4.4, no changes were observed in focal adhesions in MCF-7 cells after treatment with cordycepin 50µM.
Figure 4.4: Immunohistochemistry of MCF-7 cells treated with cordycepin:
On coverslips MCF-7 cells were seeded the day before treatment. Next day cells were treated for 3 hours with cordycepin (50µM). The cells were fixed and stained with an antibody against vinculin (red) to detect focal adhesions. Images were taken at 63X magnification.
4.5 Discussion
In this chapter we have shown that like in ASM cells, gene expression of inflammatory mRNAs (CXCL1 and IL8) in MCF-7 cells are sensitive to cordycepin. However control mRNAs which are more abundant and stable (RPL10A and ACTB) remain unchanged. This indicates that effects on inflammatory mRNAs by cordycepin are not cell type specific but seem to be gene type specific. Cordycepin
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10µM already inhibits polyadenylation compared to 50µM which clearly indicates that cordycepin represses polyadenylation in MCF7 cells. Therefore it can be concluded that as in ASM cells, cordycepin also inhibits the gene expression of several inflammatory mRNAs in MCF-7 cells. TWIST1 was also found to be downregulated in MCF-7 cells, same as previously observed in MCF-10A cells.
Pentostatin, an adenosine deaminases inhibitor, enhances the potency of cordycepin, presumably by preventing its degradation and improves its efficacy over time. We demonstrate that at the 2 and 8 hour time points Polyadenylation is equally affected by cordycepin in the absence or presence of pentostatin however poly(A) tail size recovers after 24 hours in the absence of pentostatin. The lack of recovery in the cells treated with cordycepin and pentostatin is likely to be due to the death of the cells by apoptosis (Richa Singhania, personal communication). We have previously seen effects on cell adhesion in NIH3T3 cells, but in MCF-7 cells no changes in focal adhesions were detected by immunohistochemistry. We did observe changes in cell shape under the microscope in tissue culture dishes, but these changes seem to have vanished when we plated the cells on glass coverslips to perform immunohistochemistry in detail. This might be due to the difference in the adhesion surface, the seeding density or the time in culture after seeding. These parameters may have to be re-examined in the future.
5 Investigating the mechanism of action of
cordycepin in MCF-7
In this chapter I will examine the mode of action of cordycepin in detail in MCF-7 cells. As observed previously, the stability of cordycepin improves when used in combination with pentostatin over time, however adverse effects of pentostatin have been reported. Other adenosine analogues which inhibit polyadenylation might therefore be better drugs than cordycepin. Adenosine analogues with their mode of action and further details were listed in table 8. However, we will have to determine if such compounds have similar mechanisms of action to cordycepin by
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also examining their effects on protein synthesis, 4EBP phosphorylation and effects on inflammatory mRNAs and polyadenylation, which could be more stable than cordycepin.
Table 8: Description of adenosine analogues and related compounds:
Chemical structure, mode of action and biological effects were summarized for all examined adenosine (ado) analogues and related compounds (Metformin).
Adenosine
analogues Chemical Structures Biological effects Mode of action
Cordycepin
Medicinal properties of cordycepin range from anti- bacterial and anti-fungal to anti-malarial, antivirus, anti- harpes, anti-leukemic and anti- diabetic (Shin et al. 2009). Pharmacological activities such as anti-metastatic, anti-oxidant and immunomodulatory effects by suppression of over expressed inflammation (Jeong et al. 2010). Antiapoptotic reduces cell proliferation and metastasis, inhibits cell signalling, pathways (He et al. 2010) and have anti-tumor activity in colon, leukemia, bladder, and lung carcinoma cells (Yoshikawa et al. 2008). Lipid peroxide formation inhibition (Buenz et al. 2005).
Premature termination of chain by inhibiting polyadenylation (Kondrashov et al; 2012)
8 Bromo ado In multiple myeloma cells,
mantle cell lymphoma, breast cancer cell lines and in primary CLL lymphocytes decline in ATP pool observed (Dennison et al. 2009) Polyadenylation inhibitor, chain termination leads to inhibition of RNA synthesis (Chen et al. 2010) 8 Chloro ado 8 Amino ado 8 Azido ado C8 Ado X=Br,Cl,NH2,N3
110 DRB or 5,6-Dichloro- 1- -D- ribofuranosyl benzimidazole
Induce DNA damage pathway leads to arrest at G1 phase in cancer cells (Turinetto et al. 2009). Affects polyadenylation by reducing the recruitment of polyadenylation factors to RNA polymerase II(Glover-Cutter et al. 2008) , act as inhibitors of transcriptional CDK7 and CDK9 the kinases involves in phosphorylation of C-terminal domain (CTD) of RNA polymerase II (Chen et al. 2009, Turinetto et al. 2009, Mapendano et al. 2010).
Se13 and Se15 Unknown (gifted) May act same as DRB cdk9 inhibitor
could interfere in polyadenylation Cladribine or 2-Chloro- - deoxyadenosin e
Used in hairy cell leukemia, cell cycle arrest and induces apoptosis
Inhibits DNA repair, DNA synthesis and aggregation of DNA strand breaks (Robak and Robak 2012). Interferes in RNA synthesis, also inhibits a poly(A) polymerase activity (Chen et al. 2008) -Deoxy- - (methylthio) ado
Act as an anticancer agent (Christopher et al. 2002, Avila et al. 2004), inhibitor of cell proliferation (Christa et al. 1984) By-product of polyamine metabolism, 5'- methylthioadenosi ne phosphorylase (MTAP) a tumor suppressor gene involved in purine salvage pathway. (Christopher et al. 2002, Bertino et al. 2011)
111 Metformin or 1,1-Dimethylbi guanide hydrochloride
Anticancer drug in animals used in bowel cancer, anti diabetic, lower the chance of cancer in patients suffering with type 2 diabetics (Libby et al. 2009, Martin-Castillo et al. 2010, Loubiere et al. 2013). AMPK activator (Dowling et al. 2007), similar cellular effects to cordycepin.
If cordycepin affects cells primarily by its properties as a polyadenylation inhibitor, unrelated compounds that affect polyadenylation should have similar effects. We therefore also investigated the effect of siRNA knockdown of different PAPs on total poly(A) content, in cell proliferation, polyadenylation and effects on gene expression of inflammatory mRNAs.