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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.
Anticancer Agents Med Chem. 2018; 18(9):1330-1337.AA

Abstract

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.

Authors+Show Affiliations

Laboratory of Molecular Toxicology, Faculty of Natural and Life Sciences, University of MSB-Jijel, Jijel 18000, Algeria.Laboratory of Molecular Toxicology, Faculty of Natural and Life Sciences, University of MSB-Jijel, Jijel 18000, Algeria.Laboratory of Molecular Toxicology, Faculty of Natural and Life Sciences, University of MSB-Jijel, Jijel 18000, Algeria.Department of Pharmacology, School of Medicine, The University of Jordan, Amman 11942, Jordan.Department of Physiology and Biochemistry, School of Medicine, The University of Jordan, Amman 11942, Jordan.Hamdi Mango Center for Scientific Research, The University of Jordan, Amman 11942, Jordan.Department of Physiology and Biochemistry, School of Medicine, The University of Jordan, Amman 11942, Jordan.Department of Pharmacology, School of Medicine, The University of Jordan, Amman 11942, Jordan.Department of Pharmacology, School of Medicine, The University of Jordan, Amman 11942, Jordan.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

30088453

Citation

Kebsa, Wided, et al. "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." Anti-cancer Agents in Medicinal Chemistry, vol. 18, no. 9, 2018, pp. 1330-1337.
Kebsa W, Lahouel M, Rouibah H, et al. 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. Anticancer Agents Med Chem. 2018;18(9):1330-1337.
Kebsa, W., Lahouel, M., Rouibah, H., Zihlif, M., Ahram, M., Abu-Irmaileh, B., Mustafa, E., Al-Ameer, H. J., & Al Shhab, M. (2018). 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. Anti-cancer Agents in Medicinal Chemistry, 18(9), 1330-1337. https://doi.org/10.2174/1871520618666180808100800
Kebsa W, et al. 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. Anticancer Agents Med Chem. 2018;18(9):1330-1337. PubMed PMID: 30088453.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Kebsa,Wided, AU - Lahouel,Mesbah, AU - Rouibah,Hassiba, AU - Zihlif,Malek, AU - Ahram,Mamoun, AU - Abu-Irmaileh,Bashaer, AU - Mustafa,Ebtihal, AU - Al-Ameer,Hamzeh J, AU - Al Shhab,Mohammad, PY - 2018/01/26/received PY - 2018/07/07/revised PY - 2018/07/17/accepted PY - 2018/8/9/pubmed PY - 2019/6/27/medline PY - 2018/8/9/entrez KW - Multi-drug resistance KW - P-glycoprotein KW - adenocarcima (A549/DOX) cells KW - apoptosis KW - cell cycle KW - propolis. SP - 1330 EP - 1337 JF - Anti-cancer agents in medicinal chemistry JO - Anticancer Agents Med Chem VL - 18 IS - 9 N2 - 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. SN - 1875-5992 UR - https://www.unboundmedicine.com/medline/citation/30088453/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_ L2 - http://www.eurekaselect.com/164448/article DB - PRIME DP - Unbound Medicine ER -