2) Given that E6 inactivates p53-dependent pathways, cervical cancer cells are p

Question

2) Given that E6 inactivates p53-dependent pathways, cervical cancer cells are particularly resistant to traditional treatments such as chemotherapy, the search is ongoing for new alternative therapeutics to be used in conjunction with current chemotherapeutic drugs. One such candidate is the plant flavonoid Luteolin. In 2014, Ham et al evaluate the effectiveness of Luteolin on sensitizing cervical cancer cells to apoptosis.

f) The authors sought to further verify extrinsic and intrinsic apoptotic pathway activation by looking at levels of FAS, FADD, Bcl-2, and cytoplasmic cytochrome C (As shown in Figure 3). To which pathway, extrinsic or intrinsic, do each of these proteins belong?

Fas -

FADD -

BCL-2 -

Cytochrome C –

i. What is the relationship between Fas and FADD?

ii. What role does BCL-2 play in apoptosis?

iii. Define Bcl-2 as a tumor suppressor or a protooncogene.

iv. Why did the authors choose to look at cytoplasmic cytochrome C as a measure of apoptosis?

Figure 3: Ham et al A Fas Fas FADD Luteolin (UM) 3 10 - 20 Bcl-2 Fold change Fold chang . Luteolin (UM) 5 10 20 - Total Cytc Cytosol Cytc 0 + - O 20 O 20 Luteolin (UM) Luteolin (UM)

Explanation / Answer

Fas or First apoptosis signal receptor is a cell surface receptor that initiates apoptosis and hence is part of the extrinsic pathway.

FADD or Fas- associated protein with death domain is an adaptor protein that along with Fas protein causes apoptosis and hence is also part of the extrinsic pathway.

Bcl-2 or B-cell lymphoma 2 is a regulatory protein found in B-cells that can either induce or initiate apoptosis and hence is part of the intrinsic pathway.

Cytochrome C is a mitochondrial protein that is generally bound to lipid molecule called cardiolipin. During apoptosis, this protein dissociates from cardiolipin and is found in the cytoplasm of cells. Therefore this protein belongs to the intrinsic pathway.

i) Fas and FADD proteins are members of the death-inducing signalling complex that is formed during apoptosis.

Upon stimulation by the Fas ligand, the Fas receptor trimerises and causes the binding of FADD protein to the cytoplasmic region of the Fas repcetor. This binding event results in the recruitment of procaspases and leads to the formation of a complex known as the death inducing signalling complex (DISC). In the DISC, inactive procaspases are activated and cleave intracellular proteins, which ultimately leads to apoptosis of the cell.

ii) Bcl-2 or B-cell lymphoma 2 is a protein that is found in the outer membrane of the mitochondria and is involved in regulating mitochondiral dynamics as well as mitichondrial fission and fusion. They also regulate the ROS production in mitochondria by permeabilizing the membrane. Increase in ROS results in release of cytochrome C into the cytoplasm and thus initate the apoptosis cascade.

iii) Bcl-2 plays a key role in cancer. It is reported that damage to this gene results in a number of different kind of cancer. Over expression of this protein in, which is an anti apoptotic protein along with a protooncogene results in cancer. Hence this pritein ca nbe defined as a tumor suppressor.

iv) Cytochrome C is a very key enzyme that is part of the ETC and is found only in the inner membrane of the mitochondria. Ususally this protein is tightly bound to a lipid called cardiolipin.

As the cells age and ROS builds up in the mitochondria, cardiolipin gets oxidised and cytochrome C is relesed in teh cytoplasm. This released cytochrome C when reaches a threshold, is treated as a signa lfor programmed cell death or apoptosis.

Hence, due to the above mentioned reason, cytochrome C is used as a measure of apoptosis.

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