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. 2019 Apr;71(2):507-520.
doi: 10.1007/s10616-019-00297-3. Epub 2019 Feb 2.

Stress activated p38 MAPK regulates cell cycle via AP-1 factors in areca extract exposed human lung epithelial cells

Rashmi Nagesh  1 , K M Kiran Kumar  2 , M Naveen Kumar  2 , Rajeshwari H Patil  2   3 , S Chidananda Sharma  2
Affiliations

Stress activated p38 MAPK regulates cell cycle via AP-1 factors in areca extract exposed human lung epithelial cells

Rashmi Nagesh et al. Cytotechnology. 2019 Apr.

Abstract

Areca nut chewing habits are associated with several oral manifestations like leukoplakia, submucous fibrosis and oral squamous cell carcinoma. Although numerous evidence on areca toxicity is known but the mechanistic pathway of disease causation is to be studied. Aqueous areca nut extract treated A549 cells showed reduced cell viability by 48 h with IC50 value of 0.50%. The toxic nature of areca nut induced the production of reactive oxygen species with decreased anti-oxidant glutathione S transferase levels lead to altered redox homeostasis. PCR studies showed decreased mRNA levels of Jun and Fos AP-1 subunits on extract treatment by 48 h. The protein levels of PCNA, CDK4, RB, p53, c-Jun and c-Fos were found to be downregulated with upregulated CDK inhibitor p21 on extract treatment as compared to control. Results of FACS analysis further confirm G1/S phase cell cycle arrest on areca nut extract exposure. The regulation of downstream AP-1 subunits by MAPKs was studied by using specific inhibitors of ERK, JNK and p38 along with areca nut extract. Results showed the redox activation of MAP kinases down regulated the mRNA levels of AP-1 subunits in aqueous areca nut extract treated cells. Hence the present study aids in elucidating the role of MAP kinases in regulating the AP-1 subunits and their implications on target genes that are involved regulation of various cellular processes. Further, it would help in understanding the mechanistic aspects of the diseased state which may facilitate in designing of new therapeutic modalities that could help in cancer management.

Keywords: A549 cells; AP-1 factors; Areca nut extract; Cell cycle regulators; MAPK.

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Conflict of interest statement

Authors declare that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1
Effect of aqueous extract of areca nut on the viability of A549 cells. Values were expressed as mean ± SD (n = 4). Results were significantly different from control if *P < 0.05; **P < 0.01 and ***P < 0.001 by using student’s t-test
Fig. 2
Fig. 2
Effect of aqueous extract of areca nut on ROS production in A549 cells. Values were expressed as mean ± SD (n = 4). Results are significantly different from control if *P < 0.05; **P < 0.01 and ***P < 0.001 by using student’s t-test
Fig. 3
Fig. 3
Effect of aqueous extract of areca nut on GST activity in A549 cells. Values were expressed as mean ± SD (n = 3). Results are significantly different from control if *P < 0.05; **P < 0.01 and ***P < 0.001 by using student’s t-test. Units: µM of CDNB conjugated/min/mg protein
Fig. 4
Fig. 4
Effect of areca nut extract on induction of cell cycle arrest in A549 cells. Results were expressed as % of cells residing in different phase of cell cycle such as Sub G0, G1/S and G2/M as compared to control
Fig. 5
Fig. 5
Effect of areca nut extract on protein levels of cell cycle regulators in A549 cells. Data shown are mean ± SD (n = 3) and differences in protein levels of cell cycle regulators are statistically significant: if *P < 0.05 compared with control using one-way ANOVA followed by post hoc Tukey test. The bar graphs represent the respective densitometric analysis
Fig. 6
Fig. 6
Effect of areca nut extract on the mRNAs expression of AP-1 factors in A549 cells. Data shown are mean ± SD (n = 3) and differences in mRNA levels of AP-1 factors are statistically significant, if *P < 0.05 compared with control, as analyzed using one-way ANOVA followed by post hoc Tukey test. The bar graph represents their respective densitometric analysis
Fig. 7
Fig. 7
Effect of areca nut extract on protein levels of AP-1 factors in A549 cells. Data shown are mean ± SD (n = 3) and differences in protein levels of AP-1 factors are statistically significant: if *P < 0.05 compared with control using one-way ANOVA followed by post hoc Tukey test. The bar graph represents their respective densitometric analysis
Fig. 8
Fig. 8
Effect of U0126 on the mRNA transcripts of AP-1 factors in areca nut extract treated cells. Data shown are mean ± SD (n = 3) and differences in mRNA levels of AP-1 factors are statistically significant: if *P < 0.05 compared with control and #P < 0.05 as compared to areca nut extract values using one-way ANOVA followed by post hoc Tukey test. The bar graph represents their respective densitometric analysis
Fig. 9
Fig. 9
Effect of SP600125 on mRNA transcripts of AP-1 factors in arecanut extract treated cells. Data shown are mean ± SD (n = 3) and differences in mRNA levels of AP-1 factors are statistically significant: if *P < 0.05 compared with control and #P < 0.05 as compared to areca nut extract values using one-way ANOVA followed by post hoc Tukey test. The bar graph represents their respective densitometric analysis
Fig. 10
Fig. 10
Effect of SB202190 on the mRNA transcripts of AP-1 factors in extract treated cells. Data shown are mean ± SD (n = 3) and differences in mRNA levels of AP-1 factors are statistically significant: if *P < 0.05 compared with control and #P < 0.05 as compared to areca nut extract values using one-way ANOVA followed by post hoc Tukey test. The bar graph represents the densitometric analysis
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