Reversing Multidrug Resistance in Chemo-resistant Human Lung Adenocarcinoma (A549/DOX) Cells by Algerian Propolis Through Direct Inhibiting the P-gp Efflux-pump, G0/G1 Cell Cycle Arrest and Apoptosis Induction.

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.

Algerian Propolis Potentiates Doxorubicin Mediated Anticancer Effect Against Human Pancreatic PANC-1 Cancer Cell Line through Cell Cycle Arrest, Apoptosis Induction and P-Glycoprotein Inhibition.

BACKGROUND: Pancreatic cancer is one of the most aggressive and lethal cancers, with poor prognosis and high resistance to current chemotherapeutic agents. Therefore, new therapeutic strategies and targets are underscored. Propolis has been reported to exhibit a broad spectrum of biological activities including anticancer activity. OBJECTIVE: This study was carried out to assess the possible efficacy of Algerian propolis on the antitumor effect of doxorubicin on human pancreatic cancer cell line (PANC-1). METHODS: Modifications in cell viability, apoptosis and cell cycle progression, Pgp activity and intracellular accumulation of DOX were monitored to study the synergistic effect of Algerian propolis on the antitumor effects of DOX in PANC-1 cell line. RESULTS: Both propolis and its combination with doxorubicin inhibited cell growth in a dose-dependent manner by inducing cell cycle arrest and apoptosis. In the presence of 100 µg/ml of propolis, the IC50 of DOX against PANC-1 cells decreased by 10.9-fold. Propolis combined with DOX increased after 48h, the number of cells in the G0G1 phase with dramatical increase in sub-G1 phase to reach 47% of total cells, corresponding to an increase of senescence or apoptotic state of the cells. Dead cell assay with annexinV/PI staining demonstrated that propolis and propolis-DOX treatment resulted in a remarkable induction of apoptosis as detected by flow cytometry. It was interesting to note that propolis at its 5IC50 was found as the most potent inducer of apoptosis. Our finding revealed that induced apoptosis in our conditions was caspase-3 and caspase-9 dependent. Flow cytometry showed that propolis increased the accumulation of doxorubicin within PANC-1 cells. Moreover, fluorescent intensity detection revealed that propolis remarkably increased the retention of rhodamine-123, 7- fold compared to 3-fold of verapamil, the most effective P-gp inhibitor. CONCLUSION: In conclusion, propolis sensitize pancreatic cancer cells to DOX via enhancing the intracellular retention of DOX due to blocking the efflux activity of P-gp pump, inducing cell cycle arrest and increasing apoptosis, finding that improuve the synergism of antitumor effect of Algerian propolis and DOX in pancreatic cancer cell line. Therefore, Algerian propolis may be an effective agent in a combined treatment with doxorubicin for increased therapeutic efficacy against pancreatic cancer.

Efficiency of propolis extract against mitochondrial stress induced by antineoplasic agents (doxorubicin and vinblastin) in rats.

To assess the oxidative stress and mitochondrial dysfunction associated with disease, toxic process and aging, in vivo and in vitro preventive effect of propolis extract against mitochondrial oxidative stress induced by two anticancer drugs (doxorubicin and vinblastin) have been investigated in female wistar rat using liver and heart mitochondria. The results show that doxorubicin and vinblastin altered mitochondrial functions as observed by a decrease in respiratory control value, an activation of swelling and overproduction of superoxide anion. Myocardial tissue from doxorubicin treated rats showed a marked increase in malondialdehyde production, a depletion of reduced glutathione contents and an inhibition of catalase and superoxide dismutase activities. Similar results were also observed in liver tissue. Pretreatment of rats with propolis extract (100 mg/kg/day po) (10(-4) M ip) administered 4 days prior to doxorubicin (20 mg/kg) and/or vinblastin (2 mg/kg) injection, substantially reduced the peroxidative damage in myocardium and hepatic tissues and markedly restored the tissues catalase and SOD activities. The results strongly suggest that propolis extract protects heart and liver tissues from oxidative stress by protecting the mitochondria.