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. 2023 Jan 23;28(3):1137.
doi: 10.3390/molecules28031137.

In Vitro Anticancer Activity of Novel Ciprofloxacin Mannich Base in Lung Adenocarcinoma and High-Grade Serous Ovarian Cancer Cell Lines via Attenuating MAPK Signaling Pathway

Michael A Fawzy  1 , Rania H Abu-Baih  1 , Gamal El-Din A Abuo-Rahma  2   3 , Islam M Abdel-Rahman  3 , Azza A K El-Sheikh  4 , Maiiada H Nazmy  1
Affiliations

In Vitro Anticancer Activity of Novel Ciprofloxacin Mannich Base in Lung Adenocarcinoma and High-Grade Serous Ovarian Cancer Cell Lines via Attenuating MAPK Signaling Pathway

Michael A Fawzy et al. Molecules. .

Abstract

Novel drugs are desperately needed in order to combat a significant challenge due to chemo-therapeutic resistance and bad prognosis. This research aimed to assess the anticancer activity of a newly synthesized ciprofloxacin Mannich base (CMB) on ovarian cancer (OVCAR-3) and lung cancer (A-549) cell lines and to investigate probable involved molecular mechanisms. The cytotoxic and pro-apoptotic impact of CMB on both cell lines was investigated using MTT assay, Annexin V assay, and cell cycle analysis, as well as caspase-3 activation. Western blotting was carried out to evaluate downstream targets of the MAPK pathway, while qRT PCR was used to evaluate the gene expression pattern of the p53/Bax/Bcl2 pathway. CMB treatment showed significantly reduced cell proliferation in both OVCAR-3 and A-549 cells with half maximum inhibitory concentration (IC50) = 11.60 and 16.22 µg/mL, respectively. CMB also induced apoptosis, S phase cell cycle arrest, and up-regulated expression of p53, p21, and Bax while down-regulated Bcl2 expression. CMB also halted cell proliferation by deactivating the MAPK pathway. In conclusion, CMB may be regarded as a potential antiproliferative agent for lung and ovarian cancers due to anti-proliferative and pro-apoptotic actions via inhibition of the MAPK pathway and p53/Bax/Bcl2.

Keywords: MAPK pathway; P53/Bax/Bcl2 pathway; apoptosis; cancer; cell cycle; ciprofloxacin; drug repositioning.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Cytotoxic effects of CMB and doxorubicin treatment in OVCAR−3 and A−549 cell lines. (A) OVCAR-3 cell lines. (B) A−549 cell lines. Results from MTT test obtained after a 48−h incubation period with both treatments. Means and SE were calculated from three independent experiments.
Figure 2
Figure 2
Cell apoptosis analysis of CMB and doxorubicin treatment on OVCAR−3 and A−549 cells after incubation for 48 h. The PI staining intensity is shown on the y-axis in log units, and the FITC-Annexin V staining intensity is shown on the x-axis. Lower left (LL) quadrant of a dot or density diagram represents normal viable cells. Upper right (UR) quadrant of a dot or density map represents late apoptotic cells. Lower right (LR) quadrant of a dot or density map contains early apoptotic cells. Necrotic cells are found in the dot’s upper left (UL) quadrant. (A): OVCAR−3 and A−549 cells that have received CMB treatment (IC50). (B): OVCAR−3 and A−549 cells treated with doxorubicin (IC50). (C): OVCAR−3 and A−549 control cells with no treatment.
Figure 3
Figure 3
Cell cycle analysis of CMB and Doxorubicin treated OVCAR−3 and A−549 cells: (A): Treatment of OVCAR−3 and A−549 cells with CMB (IC50) for 48 h. (B): Treatment of OVCAR−3 and A−549 cells with doxorubicin (IC50) for 48 h. (C): OVCAR−3 and A−549 untreated control cells. (D): DNA content in each stage of cell cycle following treatment with CMB and doxorubicin (IC50) for 48 h.
Figure 3
Figure 3
Cell cycle analysis of CMB and Doxorubicin treated OVCAR−3 and A−549 cells: (A): Treatment of OVCAR−3 and A−549 cells with CMB (IC50) for 48 h. (B): Treatment of OVCAR−3 and A−549 cells with doxorubicin (IC50) for 48 h. (C): OVCAR−3 and A−549 untreated control cells. (D): DNA content in each stage of cell cycle following treatment with CMB and doxorubicin (IC50) for 48 h.
Figure 4
Figure 4
Molecular evaluation of gene expression in OVCAR-3 and A-549 cells after incubation with CMB or doxorubicin IC50 values for 24 h or 48 h. Relative gene expression is represented as mean ± SEM. (A) P53 gene expression in OVCAR-3 and A-549 cells. (B) P21 gene expression in OVCAR-3 and A-549 cells. (C) Bax mRNA level in OVCAR-3 and A-549 cells. (D) Bcl2 mRNA level in OVCAR-3 and A-549 cells. Means and SE were calculated from three independent experiments. Statistically significant results compared to untreated control were displayed as: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 5
Figure 5
Western blots of ERK1/2 protein expression in OVCAR-3 and A-549 cells incubated with CMB and doxorubicin at IC50 values for 24 h or 48 h. Means and SE were calculated from three independent experiments. Statistically significant results relative to untreated control were presented as: * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 6
Figure 6
CMB suppresses the protein expression levels of p−mkk4, p−mkk7, and p−JKN1/2. (A–D) Western blot analysis of the protein levels of p−mkk4, p−mkk7, and p−JKN1/2 in OVCAR-3 and A-549 cells, respectively, incubated with CMB and doxorubicin at their IC50 values for 24 h or 48 h. Means and SE were calculated from three independent experiments. Statistically significant results relative to untreated control were displayed as: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 7
Figure 7
Western blots of active caspase-3 levels in OVCAR-3 and A-549 cells, respectively, incubated with CMB and doxorubicin at IC50 values for 24 h or 48 h. Means and SE were calculated from three independent experiments. Statistically significant results compared to untreated control were presented as: *** p < 0.001; **** p < 0.0001.
Figure 8
Figure 8
Synthesis of novel CMB [37].
See this image and copyright information in PMC

References

    1. Amin A.R., Karpowicz P.A., Carey T.E., Arbiser J., Nahta R., Chen Z.G., Dong J.-T., Kucuk O., Khan G.N., Huang G.S. Evasion of anti-growth signaling: A key step in tumorigenesis and potential target for treatment and prophylaxis by natural compounds. Semin. Cancer Biol. 2015;35:S55–S77. doi: 10.1016/j.semcancer.2015.02.005. - DOI - PMC - PubMed
    1. Rodrigues F.C., Kumar N.A., Thakur G. Developments in the anticancer activity of structurally modified curcumin: An up-to-date review. Eur. J. Med. Chem. 2019;177:76–104. doi: 10.1016/j.ejmech.2019.04.058. - DOI - PubMed
    1. Gao T., Zhou X.-L., Liu S., Rao C.-X., Shi W., Liu J.-C. In vitro effects of nicotine on the non-small-cell lung cancer line A549. J. Pak. Med. Assoc. 2016;66:368–372. - PubMed
    1. Chi F., Wu R., Jin X., Jiang M., Zhu X. HER2 induces cell proliferation and invasion of non-small-cell lung cancer by upregulating COX-2 expression via MEK/ERK signaling pathway. OncoTargets Ther. 2016;9:2709. - PMC - PubMed
    1. Bunn P.A., Jr. Early-stage NSCLC: The role of radiotherapy and systemic therapy. J. Natl. Compr. Cancer Netw. JNCCN. 2004;2:S31–S40. - PubMed