The document summarizes a study investigating the effects of PKI-402, a dual PI3K/mTOR inhibitor, on ovarian cancer cell lines. The study used various molecular biology techniques like western blotting, RNA extraction, immunoprecipitation, and immunofluorescence to analyze the mechanism by which PKI-402 suppresses cell growth. The results showed that PKI-402 not only inhibited proliferation in cisplatin-sensitive and resistant cell lines, but also promoted degradation of Mcl-1 protein through both proteasome and autophagy-lysosome pathways. In conclusion, the different methods demonstrated the effects of inhibitors on ovarian cancer cell lines and how molecular biology techniques can evaluate cellular activity and different pathologies.

1. Isabela Quijano Caro - Soath Marcela Quijano López
Molecular Biology
III semester
Medicine

2. Introduction
Ovarian cancer generalities
Ovarian cancer mainly
affects women who have
been through the
menopause.
Ovarian cancers were previously believed
to begin only in the ovaries, but recent
evidence suggests that many ovarian
cancers may actually start in the cells in the
far (distal) end of the fallopian tubes.
Ovarian cancer is when
abnormal cells in the ovary
begin to multiply out of
control and form a tumor.
Ovarian cancer can cause
symptoms that allow its
diagnosis.

3. mTOR
Molecules Involved in Ovarian Cancer
PI3K AKT
SKOV3
A2780
PIK3CA mutated Dual PI3K/ mTOR Inhibitor
Mcl- 1 p62

4. OBJECTIVE
To investigate the mechanism by which
PKI-402 suppressed cell growth, we
observed the effects on mitochondrial
functions, mitochondrial apoptotic
pathways, and autophagy in cells.

5. Western blot assay
METODS
Were used for immune-detection
and visualization using Syngene Bio Imaging
Is an analytical technique used to identify specific proteins
in a complex mixture of proteins, such as in cell or tissue extracts.

6. RNA extraction and quantitative real-time PCR
This technique is based on enzymatic in vitro amplification. This
amplification is the geometrical increase in the number of
copies of a particular DNA sequence.
METODS
Real-time PCR was carried out for Mcl-1 using the primer
pair 5′- ATG CTT CGG AAA CTG GAC AT -3′
5′- TGG AAG AAC TCC ACA AAC CCA -3′,
for β-actin using the primer pair
5′- GAC CTG ACA GAC TAC CTC AT -3′
5′- AGA CAG CAC TGT GTT GGC TA -3′.

7. It is the technique of precipitation of an antigen protein from
solution using an antibody that specifically binds to that particular
protein.
METODS
Immunoprecipitation assay
Were used to isolate and concentrate a particular
protein from a sample containing many thousands
of different proteins.

8. METODS
Immunofluorescence assay
Immunofluorescence is a technique based on an antigen-
antibody reaction where the antibodies are tagged (labelled)
with a fluorescent dye and the antigen-antibody complex is
visualized using ultraviolet (fluorescent) microscope.
Were used to react the cells with the primary antibodies and
seconds, generating an image that allows analyzing how
strong this interaction is.

9. The inhibition of
PI3K/AKT/mTOR
signaling pathway not
only promoted the
degradation of Mcl-1
protein through the
proteasome pathway, but
also through the
autophagy
lysosome pathway.
RESULTS

10. RESULTS

11. RESULTS

12. RESULTS

13. Fulda
The PI3K/AKT/mTOR signaling pathway is involved in the regulation
of mitochondrial functions and apoptosis through a variety of mechanisms.
Discussion
Chloroquine inhibits autophagic flux by decreasing
autophagosome-lysosome fusion
C. Santiskulvong, G.E. Konecny, M. Fekete, K.Y. Chen, A. Karam, D. Mulholland, C. Eng, H. Wu, M. Song and O. Dorigo
M. Mauthe, I. Orhon, C. Rocchi, X. Zhou, M. Luhr, K.J. Hijlkema, R.P. Coppes, N. Engedal, M. Mari and F. Reggiori
Dual targeting of phosphoinositide 3-kinase and mammalian target of rapamycin using
NVP-BEZ235 as a novel therapeutic approach in human ovarian carcinoma

14. CONCLUSIONS
They found that PKI-402, a dual PI3K/mTOR
inhibitor, not only inhibited the proliferation of
cisplatin-sensitive ovarian cancer epithelial
cell line A2780, but also had a significant
inhibitory effect on cisplatin-resistant ovarian
cancer epithelial cell line SKOV3
In conclusion, the different methods used in the study
helped demonstrate how different inhibitors reacted to
ovarian cancer and their cell line
Molecular biology techniques help keep a detailed record of cellular
activity in order to evaluate different pathologies through constant
monitoring of cellular activity.
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15. Soath's Mindmup
https://app.mindmup.com/map/_free/2020/08/b06ac300e1ad11ea9be7bfff6ca22878

16. Isabela's Mindmup
https://app.lucidchart.com/documents/view/bbe133ff-58b8-40d6-b509-
b568a29c46d1/fLIdogm_myHX