Korean J Physiol Pharmacol.  2021 Jul;25(4):273-280. 10.4196/kjpp.2021.25.4.273.

Vanillin oxime inhibits lung cancer cell proliferation and activates apoptosis through JNK/ERK-CHOP pathway

• Shen J
• Su Z

• KMID: 2516867
• DOI: http://doi.org/10.4196/kjpp.2021.25.4.273

Abstract

Lung cancer despite advancement in the medical field continues to be a major threat to human lives and accounts for a high proportion of fatalities caused by cancers globally. The current study investigated vanillin oxime, a derivative of vanillin, against lung cancer cells for development of treatment and explored the mechanism. Cell viability changes by vanillin oxime were measured using MTT assay. Vanillin oxime-mediated apoptosis was detected in A549 and NCI-H2170 cells at 48 h of exposure by flow cytometry. The CEBP homologous protein (CHOP) and death receptor 5 (DR5) levels were analysed by RT-PCR and protein levels by Western blotting. Vanillin oxime in concentration-dependent way suppressed A549 and NCI-H2170 cell viabilities. On exposure to 12.5 and 15 μM concentrations of vanillin oxime elevated Bax, caspase-3, and -9 levels in A549 and NCI-H2170 cells were observed. Vanillin oxime exposure suppressed levels of Bcl-2, survivin, Bcl-xL, cFLIP, and IAPs proteins in A549 and NCI-H2170 cells. It stimulated significant elevation in DR4 and DR5 levels in A549 and NCI-H2170 cells. In A549 and NCI-H2170 cells vanillin oxime exposure caused significant (p < 0.05) enhancement in CHOP and DR5 mRNA expression. Vanillin oxime exposure of A549 and NCI-H2170 cells led to significant (p < 0.05) enhancement in levels of phosphorylated extracellular-signal-regulated kinase and c-Jun N-terminal kinase. Thus, vanillin oxime inhibits pulmonary cell proliferation via induction of apoptosis through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated pathway. Therefore, vanillin oxime may be studied further to develop a treatment for lung cancer.

Keyword

Apoptosis; Death receptors; Lung cancer; Natural products; Vanillin oxime

Figure

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  Fig. 1 Effect of vanillin oxime on A549 and NCI-H2170 cells. Vanillin oxime at 5, 7.5, 10, 12.5, 15, 17.5, and 20 µM concentrations was mixed in RPMI to determine A549 and NCI-H2170 cell viability changes at 48 h by MTT assay. MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide. *p < 0.05 and **p < 0.01 vs. control, ***p < 0.018.

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  Fig. 2 Effect of vanillin oxime on A549 and NCI-H2170 cell apoptosis. (A) Vanillin oxime at 12.5 and 15 µM concentrations was mixed in RPMI to determine A549 and NCI-H2170 cell apoptosis induction at 48 h by flow cytometry. (B) Quantification of the data obtained from flow cytometry. PI, propidium iodide. *p < 0.05 and **p < 0.01 vs. control.

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  Fig. 3 Effect of vanillin oxime on pro-apoptotic proteins. (A) Vanillin oxime at 12.5 and 15 µM concentrations was mixed in RPMI to determine cleaved caspase-3, -9 and Bax expression in A549 and NCI-H2170 cells at 48 h by Western blotting. (B) Proteins bands were quantified. *p < 0.05 and **p < 0.01 vs. control.

• 

  Fig. 4 Effect of vanillin oxime on survival proteins. Vanillin oxime at 12.5 and 15 µM concentrations was mixed in RPMI to assess survival proteins levels in (A) A549 and (B) NCI-H2170 cells by western blotting. In vanillin oxime treated cells protein expression was determined at 48 h using Western blotting assay.

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  Fig. 5 Effect of vanillin oxime on DR4 and DR5 levels. Vanillin oxime at 12.5 and 15 µM concentrations was mixed in RPMI to assess DR4 and DR5 levels in (A) A549 and (B) NCI-H2170 cells at 48 h by Western blotting. *p < 0.05 and **p < 0.01 vs. control.

• 

  Fig. 6 Effect of vanillin oxime on CHOP and DR5 levels. (A) Vanillin oxime at 12.5 and 15 µM concentrations was mixed in RPMI to assess CHOP and DR5 levels in A549 and NCI-H2170 cells at 48 h by RT-PCR assay. (B) CHOP protein bands were assayed by Western blotting. *p < 0.05 and **p < 0.01 vs. control.

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  Fig. 7 Effect of CHOP siRNA on vanillin oxime induced CHOP and DR5 levels. Vanillin oxime at 12.5 and 15 µM concentrations was mixed in RPMI to assess CHOP and DR5 levels in CHOP siRNA transfected (A) A549 and (B) NCI-H2170 cells. *p < 0.05 and **p < 0.01 vs. control.

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  Fig. 8 Effect of vanillin oxime on ERK and JNK activation. Vanillin oxime at 12.5 and 15 µM concentrations was mixed in RPMI to assess p-ERK and p-JNK levels in (A) A549 and (B) NCI-H2170 cells by Western blotting.

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  Fig. 9 Effect of ERK and JNK inhibitors on vanillin oxime induced DR5 and DR4 levels. Vanillin oxime at 12.5 and 15 µM concentrations was mixed in RPMI to assess DR5 and DR4 in SP600125 and PD98059 treated (A) A549 and (B) NCI-H2170 cells by RT-PCR. *p < 0.05 and **p < 0.01 vs. control.

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