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.

Panel A: Figure 1: Ham et al E6 1.2 Panel B: 0.8 0.6 0.4 0.2 Luteolin (uM) 5 10 20 21 20 Luteolin (HM) Luteolin (LLM 5 10 20 Cyclin A Cyclin E

a) In Figure 1, panel A, the researchers looked for an effect on E6 levels post Luteolin treatment. What does the data show (i.e. what is the effect of Luteolin on cellular levels of E6)?

b) The authors then proposed that because of the effects of Luteolin on E6, that Luteolin would sensitize cervical cancer cells to apoptosis. Do you agree with this claim? If so, why?

c) In Figure 2, panel B, the authors treated a cervical cancer cell line, HeLa, with increasing amounts of Luteolin. After 48 hours of treatment, the assayed cells via western blot for p53 and p21 as well as the S-phase Cyclins A & E.

i. Why do you think that the authors looked at both p53 and p21? I.e. what is the relationship between these two proteins.

ii. What would be 2 potential downstream effects of activating the p53-dependent pathway?

d. For each of the proteins indicate whether they showed increasing or decreasing levels with increased amounts of Luteolin.

p53

p21

Cyclin A

Cyclin E

i. Overall, the authors claimed that these results showed that cells were not proceeding into S-phase due to reactivation of p53-dependent pathways. How do these data support this claim?

Answer 1

Please find the answers below:

Answer a: According to the information, the first graph shows the effect of different concentrations of luteolin on the cellular levels of E6. Here, the researchers are interested in investigating the effect of luteolin on cell cycle arrest and hence anti-cancerous activity. Since E6 remains a negative regulator of cell cycle, this primary experiment was conducted to investigate the effect of luteolin on cellular E6 levels.

Answer b: Yes. The claim of the authors is true since the different experimental sets clearly demonstrate that luteolin treatment activates the mitochondrial caspases in the cancerous cells as well as stabilization and activation of p53-p21 cascade thus leading to apoptosis and cell cycle arrest, respectively.

Answer ci: The p53 and p21 are cellular protein regulators of cell cycle progression. The protein p21 acts downstream p53 and upon stabilization and activation, it brings about an immediate arrest in the cell cycle. Hence, in order to validate the cell-cycle regulatory and inhibitory effects of luteolin, the authors investigated the expression levels of these markers.

Answer cii: The p53 can either induce direct apoptosis by acting as a transcription factor and degrading the DNA or it can activate a down-stream signalling cascade by activating p21 and other cell-cycle dependent kinases to immediately halt cellular division.

Answer d: As the amount of luteolin increases, the cell cycle arrest and apoptosis increases, thus the effect on the positive regulators of cell cycle arrest will be demonstrated as an increase in all p53, p21, cyclin A as well as cyclin E.

Answer di: The cyclins and the p21, both are involved in cell-cycle progression at the first check-point of cell division, i.e. between S to G2 phase. Since activation of p53-p21 takes place during this phase, the claim of authors that luteolin treatment would not allow these cells to pass through the S phase are validated.