This document summarizes a study on targeting the ERK and AKT pathways in non-small cell lung cancer and triple-negative breast cancer cells with bioactives from Nigella sativa. The study found that thymoquinone (TQ) and thymol (THY) from N. sativa exhibited cytotoxic effects on cancer cells in a dose-dependent manner. Combining THY and TQ showed greater inhibition of cell viability than either component alone. Treatment with the combination also significantly downregulated expression of the AKT and ERK genes involved in proliferation. The findings suggest TQ may improve the efficacy of THY as an adjuvant therapy for lung and breast cancers.

1. “Targeting the ERK and AKT Pathways in Non-Small Cell Cancer and
Triple-Negative Breast Cancer Cells with Nigella sativa Bioactives”
YUSUF ASAD

2. • Cancer is an abnormal growth of cells which tend to proliferate in an
uncontrolled way and, in some cases, to metastasize.
• Approximately 8.2 million people die from this disease worldwide every
year.
• In lung cancer, the proliferation occurs in the tissues of lung, usually in the
cells lining air passages. The two main types are small cell lung cancer and
non-small cell lung cancer.
• In breast cancer, the cells in the breast grow out of control. The most
common type of breast cancers are ductal carcinoma and lobular carcinoma.
Introduction

3. • Cancers of the oral cavity and lungs account for over 25% deaths in
males and breast and oral cavity cancers account for 25% deaths in
females.

4. AKT Pathway
• AKT pathway is an intracellular signaling pathway, important in
apoptosis, and hence in cancer.
• Activated by IGF1 and has number of downstream effects, which either
promote protein synthesis or inhibit protein breakdown.
• In triple-negative breast cancer (TNBC) and non-small cell lung
carcinoma (NSCLC), this pathway is overactive, thus, reducing apoptosis
and allowing proliferation.

5. ERK Pathway
• The ERK cascade is under extensive
homeostatic control by feedback loops.
• Activated ERK stimulates inhibitory
phosphorylation upstream factors and
kinases, such as MEK, RAF, SOS, and
RTKs for stable cellular function.
• RAS mutants encode mutated proteins
that harbor single amino-acid substitutions
at glycine 12 (G12) and glutamine 16
(Q16) in human cancers.
• Upstream mutations lead to
hyperactivation of ERK protein,
responsible for a series of ERK signaling-
regulated substrate activation and is
consequently related to a wide range of
tumors.

6. • The Chemoprevention: kills tumor cells, doing as little damage to healthy
cells as possible (eg. cisplatin).
• Adverse effects of platinum drugs are:
Neurotoxicity
Nephrotoxicity
Ototoxicity
Acquired or ab initio resistance

7. • Nigella sativa has been used as a traditional medicine for centuries.
• The active ingredients of N. sativa have beneficial effects against many
diseases, including cancers.
• The important components are: thymoquinone (TQ), dithymoquinone
(DTQ), thymohydroquinone (THQ), and thymol (THY).

8. Aims and Objectives
The present study was conducted with the aim to examine the effects of
thymol (THY) and thymoquinone (TQ) on lung and breast cancer cell lines.
The specific objectives of the study were as follows:
• To examine the effects of Nigella sativa bioactives, THY and TQ, on lung
and breast cancer cells lines.
• To evaluate the efficacy of combination therapy with THY and TQ.
• To evaluate the effect of THY and TQ on the expression of Akt and Erk
genes in lung and breast cancer genes.

9. Methodology
Human lung epithelial L132 cells, A549, and MDA-MB-231 cells were procured
from NCCS, Pune
Toxicity Assay of CP, THY, and TQ in L132 cells by MTT
Cell Cytotoxicity Assay of single dosage of CP, THY, and TQ in A549 and MDA-MB-
231 cells
Cytotoxicity assay of combined dosage of THY and TQ in A549 and MDA-MB-231
cells

10. Methodology..
Total RNA isolation from A549 and MDA-MB-231 Cells using TRIzol Reagent
Quantification of isolated RNA
cDNA Synthesis using Verso cDNA synthesis Kit
Real-Time qRT-PCR analysis

11. Dose dependent effect of CP on cell viability in A549 and MDA-MB-231 cells by MTT assay The results
represented are the mean±S.E.M of three independent experiments performed in triplicate (***p < 0.0001)
represent significant difference compared with control) analyzed by two-way ANOVA.
1. Assessment of Cytotoxicity of A549 and MDA-MB-231 Cells Treated with CP
for 24 h.

12. Dose dependent effect of THY on cell viability in A549 and MDA-MB-231cells by MTT assay The results
represented are the mean±S.E.M of three independent experiments performed in triplicate (***p < 0.0001)
represent significant difference compared with control) analyzed by two-way ANOVA.
2. Assessment of Cytotoxicity of A549 and MDA-MB-231 Cells Treated
with TQ for 24 h.

13. Dose dependent effect of TQ on cell viability in A549 and MDA-MB-231cells by MTT assay The results
represented are the mean±S.E.M of three independent experiments performed in triplicate (***p < 0.0001)
represent significant difference compared with control) analyzed by two-way ANOVA.
3. Assessment of Cytotoxicity of A549 and MDA-MB-231 Cells Treated with
TQ for 24 h.

14. Effect of cytotoxicity of combined doses of THY+TQ in A549 and MDA-MB-231 cells . The results
represented are the mean±S.E.M of three independent experiments performed in triplicate ( ***p < 0.001
represent significant difference compared with control).
4. Assessment of Synergistic Effect of THY with TQ on the Growth Inhibition of
A549 and MDA-MB-231cells

15. Dose dependent effect of IC50 concentration of CP, THY/TQ and THY+TQ on normal human lung L132cells.
Toxic effects of CP, THY/TQ and THY+TQ on cell viability of normal L132 cells treated with different IC50
doses of CP, THY/TQ and THY+TQ for 24 h assessed by MTT assay The results represented are the
mean±S.E.M of three independent experiments performed in triplicate (*p < 0.01, **p < 0.001 represent
significant difference compared with control).
5. Assessment of the Effect of CP, THY, and TQ on the Viability of L132 cells

16. CONTROL CP THY
TQ THY+TQ
6. Evaluation of Combination Doses of THY+TQ on Nuclear Condensation in
the A549 and MDA-MB-231 Cell Lines
Effect of 24 h of exposure of CP, THY, TQ and THY+TQ on chromatin condensation in A549 cells as
measured by DAPI staining.

17. CONTROL CP THY
TQ THY+TQ
Effect of 24h of exposure of CP, THY, TQ and THY+TQ on chromatin condensation in MDA-MB-231 cells
was measured by DAPI staining.

18. Quantitative real-time RT-PCR for assessing the expression of (a) AKT in A549 and MDA-MB-231 cells
exposed to different treatments. Results are presented as mean ± SEM (n = 3); ***P<0.001, compared with
control, analyzed by one-way ANOVA (Dunnet’s multiple comparison test).
7. Effect of Combination of THY+TQ on Gene Expression of AKT using real-
time qRT-PCR in A549 cells and MDA-MB-231 cells

19. Quantitative real-time RT-PCR for assessing the expression of ERK in A549 and MDA-MB-231 cells
exposed to different treatments. Results are presented as mean ± SEM (n = 3); ***P<0.001, compared with
control, analyzed by one-way ANOVA (Dunnet’s multiple comparison test).
8.Effect of Combination of THY+TQ on Gene Expression of ERK using real-time
qRT-PCR in A549 cells and MDA-MB-231 cells

20. THY-ERK
CP-AKT
CP-ERK TQ-ERK
TQ-AKTTQ-AKT
Docked complex of CP-ERK, THY-ERK, TQ-ERK, CP-AKT, TQ-AKT and TQ-AKT. The image was obtained
by Discovery Studio 2.0

21. Conclusion
• In vitro incubation of A549 cells and MDA-MB-231 cells with combined
doses of THY and TQ showed remarkable cell death in a concentration-
dependent manner.
• Combination of THY+TQ significantly down regulated the expression of
AKT and ERK in both lung cancer and breast cancer cells.
• Our findings suggest that TQ, a natural food substance with no known
human toxicity, holds promise as an adjuvant to THY and can improve
the efficacy of THY in NSCLC and breast cancer patients.

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23. Thank you