Background:
Lung cancer is one of the most common malignancies with the highest incidence and
mortality rate worldwide. Multidrug Resistance (MDR) continues to pose a major challenge for the clinicians and
pharmacologists to effectively treat this disease. A new approach using natural substances with moderate or low
cytotoxic properties become a promising hope for reversing multidrug resistance due to pgp- overexpression.
Objective:
This study aims to explore the efficacy of Algerian propolis in reversing multidrug resistance and
sensitizing chemo-resistant lung cancer cells (A549/DOX) to chemotherapy with DOX.
Methods:
Resistant lung adenocarcinoma A549/DOX cell line was developed and used as in vitro model for
MDR. Cell viability, Annexin V-PI apoptosis assay and cell cycle progression were tested to evaluate the reversal
effect of propolis alone or in combination with DOX. Caspases 3, 8 and 9 assays were conducted to determine
the type of apoptotic pathway. To investigate the mechanisms of MDR reversal agents, intracellular accumulation
of DOX and P-gp-pump activity were investigated.
Results:
Our results showed that the obtained chemo-resistant cells were 13-fold more resistant to DOX than the
parental A549 cells. Propolis showed dramatically cell growth inhibition on A549/DOX cells (The IC50 was
50.44± 0.07µg/ml). The killing effect of propolis was due to G0/G1 cell cycle arrest and apoptosis induction.
After 24hours treatment, propolis at 100 µg/ml caused cells accumulation in G0/G1 phase and increased
with 50, 65-fold the percentage of apoptotic population sub-G. Annexin V-PI assay showed that propolis
induces apoptosis with 53.57-fold at 100 µg/ml. It induced intrinsic apoptotic pathway by increasing caspase-3
(22.15-fold) and caspase-9 (16.73-fold) activities. The direct approach to investigate the mechanisms of reversal
agents is to detect the accumulation of P-gp substrates in resistant cells. Our results indicated that resistant cells
poorly accumulated Doxorubicin and rhodamine 123 (7-fold lower) when compared to parental A549 cells,
suggesting that chemo-resistant cells overexpress P-gp which pump DOX out of cells. Propolis inhibited in a
concentration-dependent manner, the pgp efflux-pump, enhancing thereby the intracellular level of DOX with
5.48- fold against 3.33 fold obtained with verapamil, the conventional P-gp inhibitor.
Conclusion:
Taken together, Algerian propolis reverses multidrug resistance in resistant human lung
adenocarcinoma cells through direct inhibiting the transport function of pgp-pump resulting in enhancing intracellular
DOX-accumulation, G0/G1 cell cycle arrest and apoptosis induction. Thus, propolis could be developed
as a chemotherapeutic agent for reversing multidrug resistance.
Mechanisms of Induction of Cell Cycle Arrest and Cell Death by Cryptolepine in Human Lung Adenocarcinoma A549 Cells