Vapor of Volatile Oils from <em>Litsea cubeba</em> Seed Induces Apoptosis and Causes Cell Cycle Arrest in Lung Cancer Cells

<div><p>Non-small cell lung carcinoma (NSCLC) is a major killer in cancer related human death. Its therapeutic intervention requires superior efficient molecule(s) as it often becomes resistant to present chemotherapy options. Here we report that vapor of volatile oil compounds obtained from <em>Litsea cubeba</em> seeds killed human NSCLC cells, A549, through the induction of apoptosis and cell cycle arrest. Vapor generated from the combined oils (VCO) deactivated Akt, a key player in cancer cell survival and proliferation. Interestingly VCO dephosphorylated Akt at both Ser⁴⁷³ and Thr³⁰⁸; through the suppression of mTOR and pPDK1 respectively. As a consequence of this, diminished phosphorylation of Bad occurred along with the decreased Bcl-xL expression. This subsequently enhanced Bax levels permitting the release of mitochondrial cytochrome c into the cytosol which concomitantly activated caspase 9 and caspase 3 resulting apoptotic cell death. Impairment of Akt activation by VCO also deactivated Mdm2 that effected overexpression of p53 which in turn upregulated p21 expression. This causes enhanced p21 binding to cyclin D1 that halted G1 to S phase progression. Taken together, VCO produces two prong effects on lung cancer cells, it induces apoptosis and blocked cancer cell proliferation, both occurred due to the deactivation of Akt. In addition, it has another crucial advantage: VCO could be directly delivered to lung cancer tissue through inhalation.</p> </div

VCO induces apoptosis in A549 lung cancer cells.

<p>(<b>A</b>) Annexin-Cy3 (red) and 6-CFDA (green) double staining of apoptotic cells was examined by fluorescence microscopy where VCO treated A549 cells showed both green and red stains and control (untreated) cells stained green only. (<b>B</b>) Percentage of apoptotic A549 cells was measured at different time points (0 h, 12 h, 24 h, 36 h) with VCO treatments. (<b>C</b>) Mitochondrial membrane potential was observed in control and VCO exposed (36 h) A549 lung cancer cells by JC-1 staining assay. (<b>D</b>) Apoptotic DNA fragmentation was observed by VCO treated A-549 cells on 1.5% agarose gel electrophoresis. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01 versus control (0 h). Bar represents 20 µm.</p

Deactivation of Bad with altered Bcl-xL/Bax ratio on mitochondrial membrane by VCO exposure.

<p>(<b>A</b>) Immunoblot analysis was performed to evaluate the level of pBad Ser¹³⁶ and Bad in A549 cells exposed with VCO for different time periods (0 h, 12 h, 24 h, 36 h). β-actin served as internal control. Bands were quantified by densitometric analysis where pBad level was compared with Bad level. (<b>B</b>) Protein level of Bcl-xL and Bax of these cells were also evaluated by immunoblot analysis. Densitometric analysis showed Bcl-xL was negatively correlated with Bax level when A549 cells were exposed with VCO. Values are means ± SEM of three independent experiments, *p<0.05, **p<0.01 versus control (0 h).</p

Time dependent inhibition of Akt phosphorylation by VCO.

<p>(<b>A</b>, <b>B</b>) Immunoblot analysis of Akt phosphorylation at Thr³⁰⁸ (A) and Ser⁴⁷³ (B) in A549 treated cells with VCO for the indicated time period (upper panel). Fold change represents the protein level of the VCO treated cells relative to the control cells. Bands were quantified by densitometric analysis where pAkt level was then normalized to the total Akt level (lower panel). β-actin served as loading control. (<b>C</b>, <b>D</b>) Immunoblot analysis of pPDK1 Ser ²⁴¹ (C) and mTOR (D) was done at different time hour (0 h, 12 h, 24 h, 36 h) exposure of VCO to A549 cells (upper panel). Bands were quantified by densitometric analysis where pPDK1 or mTOR level was then normalized with β-actin which is represented by folds change (lower panel). Figures are representative of three independent experiments, *p<0.01, **p<0.001 versus control (0 h).</p

Effect of VCO on the viability of A549 cells by MTT assay.

<p>(<b>A</b>) Cell viability of A549 lung cancer cells were measured when exposed to vapors of different dilutions (10⁶ to 10²) of crude oil for 72 h by using MTT assay and the data was expressed as % of cell survivability relative to control. (<b>B</b>) Chemical structures of four most available compounds (C1- Citronellal; C2- neo-isopulegol; C3- isopulegol; C4- citronellol) isolated from <i>Litsea cubeba</i> seed essential oil. (<b>C</b>) Percentage of cell death was observed when A549 cells were exposed individually with these compounds for 72 h. (<b>D</b>) Effect of VCO (C2∶C3∶C4 as 1∶1∶1) and C4 on cell death at 72 h was observed by MTT assay, which was visualized by microscopic images. (<b>E</b>) Cell survivability was measured at different time intervals (24, 48, 72 h) with VCO exposure on A549 cells. (<b>F</b>) Western blot of Akt phosphorylation at Thr³⁰⁸, Ser⁴⁷³ and total Akt in A549 cells treated without (Con) with C1, C2, C3, C4 and VCO for 36 hours. β-actin served as internal loading control. Values are means ± SEM of 3 individual experiments. *p<0.05, **p<0.01 versus control and #p<0.05 versus C4.</p

VCO induces apoptotic cell death by activating caspase cascade.

<p>(<b>A</b>) A549 cells were exposed with VCO for 36 h followed by staining of mitochondria with Mitotracker (red) and cytochrome c with FITC conjugated anti-cytochrome c antibody (green). (<b>B</b>) Immunoblot analysis was done by using anti-cleaved caspase-9 or caspase-3 antibodies in A-549 cells incubated in the presence of VCO at 0 h, 24 h, 36 h time intervals. β-actin used as internal control. (<b>C</b>) A549 cells were exposed with VCO for indicated time periods and on termination of exposure, cells were lysed and caspase 3 activity was measured in DTX multimode detector by using proluminescent caspase 3 as the substrate. (<b>D</b>) PARP cleavage was observed in VCO exposed cells by immunoblot analysis using anti-PARP antibody. β-actin used as loading control. Values are means ± SEM of three independent experiments, *p<0.01, **p<0.001 versus control (0 h). Bar represents 20 µm.</p