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

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

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

In streptozotocin induced diabetic mice dmp acts like insulin.

<p>dmp activates insulin signaling in STZ mice. STZ induced BALB/c mice were starved for 12 h followed by oral administration of dmp (300μg kg^-1 bw) or vehicle. (A) Blood glucose level was detected at different times. *<i>p</i>< 0.05 <i>versus</i> STZ (vehicle), **<i>p</i>< 0.01 <i>versus</i> STZ (vehicle). (B) 4 h after dmp treatment, skeletal muscle tissues were collected from BALB/c mice (Con) or STZ mice or STZ mice fed with dmp, lysed and subjected to immunoblot using anti-pIR and anti-IR antibodies. (C) [¹⁴C] 2-DOG uptake (top) and [³H] Fatty acid uptake (bottom) by skeletal muscle or adipose tissue from above mentioned mice were determined in a liquid scintillation counter. *<i>P</i><0.05 <i>versus</i> Con; #<i>P</i><0.05 <i>versus</i> STZ (vehicle). (D) Skeletal muscle from STZ mice was incubated with insulin (100 nM) or dmp (250 nM) and [¹⁴C] 2-DOG uptake was measured at different time intervals. *<i>P</i><0.05 or **<i>P</i><0.01 <i>versus</i> STZ (vehicle). (E) dmp was orally administered to BL6 mice (5mg kg^-1 bw) and plasma dmp level was measured at different time intervals. All values are represented as mean ± s.e.m. (n = 5).</p

Interaction between dmp and IR.

<p>(A) Binding of recombinant insulin with insulin receptor (IR) was studied by Surface Plasmon Resonance (SPR) using varied concentrations of insulin. (B) Binding of dmp to IR was studied by SPR where increasing concentrations of dmp from 1–50 μM were flowed over immobilized IR protein on CM5 chips. Binding affinity of dmp to IR is represented by KD value (1.17 μM). Rmax value is 8.880 (RU) which represents maximum binding capacity of dmp with IR. (C) Fluorescence spectra of IR-dmp. All steady-state fluorescence measurements were carried out using an excitation wavelength of 280 nm. The emission spectra were traced from 300 to 500 nm. The concentration of IR was 0.3μM whereas varied concentrations of dmp was used for fluorescence spectra, it was gradually increased from 0–0.6μM. Binding constant was calculated from Stern–Volmer equation Io/I = 1+Ksv [Q] [dmp]. Quenching constant was Xhalf = 1.0118 μM calculated accordingly where Io and I are fluorescence intensities in the absence or presence of the quencher (dmp) respectively and Ksv was quenching constant.</p

dmp binding to IR augments insulin signalling pathway.

<p>(A) dmp fails to activate EGFR. L6 myotubes or 3T3L1 adipocytes were treated with or without 250 nM dmp for 4h. The cell lysates were analyzed by immunoblotting with anti-pEGFR and anti-EGFR antibodies. (B,C) dmp can induce IR phosphorylation in a dose dependent manner. L6 myotubes were treated with insulin (20–120 nm) or dmp (50–300 nm) for 4h and IR phosphorylation was monitored by ELISA (B) or immunoblotting with anti-pIR and anti-IR antibodies (C). (D) IR kinase activity was determined in L6 myotubes which were incubated with varied concentrations of insulin or dmp. (E) dmp stimulates IR and its downstream kinases phosphorylation. L6 myotubes or 3T3L1 adipocytes were treated with or without Insulin (100 nM) or dmp (250 nM) for 4h and the IR phosphorylation and its downstream signalling were monitored by immunoblotting. (F) L6 myotubes transfected with GFP-GLUT4 chimeric gene were incubated with insulin (100 nM) or dmp (250nM) for 4h. Cells on the cover slips were fixed in paraformaldehyde and observed under florescent microscope for GFP-GLUT4 translocation. (G) dmp like insulin promotes glucose uptake. L6 myotubes or skeletal muscle cells from soleus muscle of neonatal mice (2-3days) were incubated with 100 nm insulin or 250 nm dmp for 25 min. [¹⁴C] 2-DOG was then added, and the cells were further incubated for 5 min. [¹⁴C] 2-DOG uptake was measured by scintillation counting. *<i>P</i><0.05 <i>versus</i> Con; **<i>P</i><0.01 <i>versus</i> Con. (H) dmp augments fatty acid uptake. Primary culture adipocytes or 3T3L1 adipocytes were treated with 100 nm insulin or 250 nm dmp for 4h followed by incubation with [³H] Palmitate for 15 min. [³H] Palmitate uptake was measured in a liquid scintillation counter. *<i>P</i><0.05 <i>versus</i> Con; **<i>P</i><0.01 <i>versus</i> Con. (I) L6 myotubes were tranfected with IR siRNA(IR^KD) followed by estimation of IR gene and protein levels by qPCR (left) and immunoblotting (right) respectively. *<i>P</i><0.05 <i>versus</i> Con. (J) IR^KD L6 myotubes were incubated with dmp for 4h and [¹⁴C] 2-DOG uptake was measured according to the above description. **<i>P</i><0.01 <i>versus</i> Con. All values are represented as mean ± s.e.m. (n = 5).</p

dmp improves energy homeostasis in <i>db/db</i> mice.

<p>(A) BL6 mice were orally administrated with vehicle or dmp (300 μg kg^-1 bw) for 28 days. Body weight was recorded on the days mentioned in the figure. (B) <i>db/db</i> mice were orally administered with vehicle or dmp (300 μg kg^-1 bw) for 28 days. Weight of the abdominal fat was recorded. (C) Food intake was estimated in <i>db/db</i> (vechile) and dmp fed <i>db/db</i> mice. (D) Metabolic activities were measured by indirect colorimetry in BL6, <i>db/db</i> (vechile) and dmp fed <i>db/db</i> mice during day and night periods. The experimental animals were placed in metabolic cage and average hourly oxygen consumption (V̇O₂) and carbon dioxide production (VCO₂) were measured. Accordingly, RER and energy expenditure (EE) were calculated. *<i>P</i><0.05 <i>versus</i> BL6; #<i>P</i><0.05 <i>versus db/db</i> (vechile). (E) Western blot showing phosphorylation status of pAMPK and AMPK in the skeletal muscle of BL6, <i>db/db</i> (vechile) and dmp fed <i>db/db</i> mice. (F) Immunoblots showing abundance of PGC1α, NRF1 and tfam level in dmp treated mice. (G) Total DNA was extracted from muscle tissue and the content of mtDNA was calculated using real-time quantitative PCR by measuring the threshold cycle ratio (㥆Ct) of a mitochondrial-encoded gene COXII versus a nuclear encoded gene RIP140. *<i>P</i><0.05 <i>versus</i> BL6; #<i>P</i><0.05 <i>versus db/db</i> (vechile). (H) Complex IV activity (top) and ATP production (bottom) was measured in mitochondria isolated from skeletal muscle of BL6, <i>db/db</i> (vehicle) and dmp fed <i>db/db</i> mice. *<i>P</i><0.05 <i>versus</i> BL6; #<i>P</i><0.05 <i>versus db/db</i> (vechile). (I) <i>db/db</i> mice were orally administrated with vehicle or dmp (300 μg kg bw^-1) for 28 days. Blood glucose concentration (GTT) was measured before and after oral gavages of 1g glucose kg bw^-1 at the indicated time points. ITT was performed after injecting mice with 0.7U insulin kg bw^-1. Fasting insulin and fasting glucose was estimated and HOMA-IR was calculated. #<i>P</i><0.05 <i>versus db/db</i> (vechile). All values are represented as mean ± s.e.m. (n = 5).</p