Clinical treatment

Depending on the stage of the disease and other factors, the main treatment options for people with NSCLC include: surgery,

radiofrequency ablation, radiation therapy, chemotherapy, targeted therapies and immunotherapy [30]. Anti-cancer chemotherapy treatment were done by injecting drug into a vein or taken by mouth. The drugs enter the bloodstream and go throughout the body, making this treatment useful for cancer anywhere in the body [30, 31].

The chemotherapy drugs most often used for NSCLC include:

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Cisplatin, Carboplatin, camptothecin analogs, Paclitaxel (Taxol), Albumin-bound paclitaxel (nab-paclitaxel, Abraxane), Docetaxel

(Taxotere), Gemcitabine (Gemzar), Vinorelbine (Navelbine), Irinotecan (Camptosar), Etoposide (VP-16), Vinblastine and Pemetrexed (Alimta) [32, 33]. The camptothecin analogs, blocks DNA supercoils by inhibiting DNA topoisomerase I [34].

Gemcitabine, a nucleoside analog, represents one of the building blocks of nucleic acids [35]. Platinum agents induce DNA cross-linking and apoptotic cell death through binding with DNA. The cisplatin is currently used in lung cancer patients The drug was linked with DNA and inhibited cell growth, thus lead to cell death. In addition there are target therapies for gene mutation patients [36]. Iressa (Gefitinib) is the first selective inhibitor of EGFR tyrosine kinase domain. Taxol (Paclitaxel) is approved in the UK for treating ovarian, breast and lung, bladder, prostate, melanoma, esophageal, and other types of solid tumor cancers as well as Kaposi's sarcoma [37, 38]. Paclitaxel induces cell type-dependent p53, p21 and G1/G2 arrest, thus inhibits mitosis and induces apoptosis [39-41].

In this dissertation, we focused on the role of p53 and its relationship with cancer therapy agent EMMQ.

11. Novel indolylquinoline derivative

The indolylquinoline derivative contains both indole and quinoline subunits. Anti-leishmanial activities were found with indolylquinoline derivatives. Leishmaniasis is a disease complex caused by 17 different

13

species of protozoan parasites belonging to the genus Leishmania [42].

Pentamidine and amphotericin B have been used as treatment of leishmaniasis for many years. Recently reports demonstrated the

antileishmanial activity of the indolylquinoline derivatives both in vitro and in vivo [43].

12. Aims of the study

The study is to determine if EMMQ is a potential alternative to treat human lung cancer cells byexamining the growth rate of cells and

xenograft tumors.

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IV. MATERIALS AND METHODS 1. Chemicals and cell culture

The synthetic compound, EMMQ MW 298.4, the structure shown in Fig 1A) [44], was purified to more than 98% purity and dissolved in DMSO at 10 mM for storage. Three cell lines, H460 (HTB177^TM), H1299 (CRL5803^TM) and A549 (CCL185^TM) of human NSCLC cells and

MRC-5 of human Lung fibroblast cells (CCL-171 ^TM) were acquired from ATCC and maintained in DMEM and cultured with L-glutamine, sodium pyruvate, and supplied with 10% heat-inactivated FBS under 5% CO2 at 37 °C. The selected stable H1299 clones transfected with

cytomegalovirus promoter-driven pcDNA-p53 encoding full-length wild type p53 (H1299/p53) or mutant p53R267P (H1299/p53^R267P) were maintained in 10 % serum-supplemented DMEM. Short hairpin RNA targeting p53 and scrambled non-specificity was acquired from the National RNAi Platform, Academia Sinica, Taipei, Taiwan.

2. Cell viability assay

The cell cytotoxicity was measured as previously described [45] . Briefly, cells were cultured at 1.5×10³ cells per well in 96-well plates.

The attached cells supplemented with minimal amounts of FBS were treated with different concentration of cisplatin (Sigma), EMMQ or vehicle control DMSO at 37°C for 48 h. Cells were then kept in 10 μL of MTT (5 mg/mL) or cisplatin with medium at 37 °C for 4 h. MTT was then removed, added with 100 μL DMSO into each well and the

absorption measured by measuring the absorbance at 570 nm wavelength

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with a 96-well microplate reader (Thermo Fisher Scientific, USA).

3. Colony forming assay

Cells were seeded at 50 cells per well in a 6-well plates for 16 h to allow attachment and then added with EMMQ at different concentrations or vehicle control for 48 h. After replacing with fresh media and growing in 3 mL medium for 14 days, the colonies were fixed before staining with 0.5% methylene blue in ethanol for 4 h. The size and number of stained colonies with more than 50 cells were counted under inverted phase contrast microscope. Colony formation was calculated as a percentage to untreated control cultures.

4. Comet assay

Conventional slides were covered with a layer of 70 μL 0.5 % normal agarose and 0.5% low melting point agarose (GIBCO-BRL). A volume of 70 μL of low melting point agarose (0.5%, w/v) (GIBCO-BRL) was mixed with approximately 2 × 10⁴ cells suspended in 15 μL of media.

The mixture was then layered onto the slides, and immediately overlaid with coverslips. After agarose solidification at 25 °C for 30 min, the coverslips were removed and the slides immersed 60 min at 4 °C in fresh lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10 and 1% Triton X-100). The slides were soaked in the alkaline buffer (300 mM NaOH and 1 mM Na₂EDTA at pH 13) on the ice bath for 20 min and electrophoresed (30 V and 300 mA) for 20 min. Slides were then

transferred to the neutralization buffer (0.4 M Tris-HCl at pH 7.5) on ice

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bath for 15 min. Finally, slides were dried in methanol for 5 min and stained with 50 μL of PI (4 µg/mL). The tails were observed under a fluorescence microscope (Nikon, Japan) and quantified by using CometScore™ software (Tritek Corp, Sumerduck, VA).

5. Determination of apoptosis

Double staining with Annexin V-FITC and PI

Cells were seeded at 2 ×10⁵ cells per well in 12-well plates and allowed to attach overnight. Cells were treated by various concentrations of EMMQ incubated at 37 °C for 48 h. DMSO containing medium served as the vehicle control. The cells were trypsinized and stained with1 μL annexin-V/FITC (20 µg/mL, BD Bioscience) and 1 μL of PI (50 µg/mL) at room temperature for 30 min in the dark. The early and late phase of apoptosis was measured by annexin V-FITC/PI Apoptosis Detection Kit (BD Bioscience). The flow cytometer FACS CaliburTM (BD Bioscience) was used for analysis and the data were analyzed using the FlowJo

software (Tree Star, Inc.).

Cell-cycle distribution

Cell cycle distribution was determined by suspending cells in 70 % ethanol and kept at -20 °C for 24 h prior to addition of 10 µg/mL of PI (Sigma, St. Louis, MO) and 10 µg/mL of RNaseA (ICN Pharmaceutical;

Costa Mesa, CA) in PBS (UniRegion Bio-tech, Taiwan) for 30 min. The data as acquired by flow cytometry were analyzed with software FlowJo (Tree Star, Inc.).

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6. Determination of ΔΨm

ΔΨm was determined using MitoPT™ JC-1 Assay Kit

(ImmunoChemistry Technologies, Bloomington, USA). Briefly, the seeded cells were cultured in 2% serum-supplemented DMEM containing different concentration of EMMQ or vehicle control and incubated at 37

°C for the time points as indicated. The collected cells were washed with 1× assay buffer. After centrifugation at 1,000 rpm for 5 min, cell pellets were stained with 250 μL mixture containing 5 μL of JC-1 with 995 μL 1× assay buffer for 25 min at 37 °C. The residual JC-1 was removed by centrifugation at 1,000 rpm for 5 min and the pellet mixed with 1× assay buffer. JC-1 fluorescence was measured to assess the emission shift from green (530 nm) to red (590 nm) using 488 nm excitation wavelength.

Data were given as the relative ratio of green to red fluorescence intensities, indicating the level of depolarization of the mitochondrial membrane potential. The data as determined by FACS CaliburTM were quantified and the expressed as the percentage of mitochondrial

membrane potential drop relative to those of untreated cells.

7. Release of cytochrome c release

The harvested cells after treatment were treated with 100 μL

digitonin (50 µg/mL PBS, 100 mM potassium iodide and 1 mM EDTA) for 5 min on ice until more than 95 % of cells permeabilized. Cells were then fixed and stained with 3.7 % formaldehyde and DAPI (1:3,000) (Sigma, USA) in PBS for 20 min at room temperature, washed thrice in

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PBS, and incubated in blocking buffer (3% bovine serum albumin (Themo, USA) and 0.05 % saponin in PBS) for 1 h. The cells were incubated overnight at 4°C with anti-cytochrome c mouse monoclonal antibody (BD PharMingen) that was diluted to 1:200 in blocking buffer, washed thrice, and incubated for 1 h at room temperature with

TRITC-conjugated goat anti-mouse (Santa Cruz) in blocking buffer. The cells were then counterstained with Mitotracker Green (Invitrogen Life Technologies). The samples were detected using a Leica TCS SP5 Confocal Spectral Microscope.

8. Western blot analysis

The western blot analysis was determined by electrophoresis of the protein contents of cell lysates were collected and the concentrations quantitated using BCA assay (Pierce Biotechnology, Rockford, IL). A total of 20 µg of protein were resolved by electrophoresis through

SDS-PAGE gel was transferred to nitrocellulose filters, blocked with 5%

of Skim Milk (BD, Mansfield, MA) and incubated with primary and secondary antibodies. The emitted chemiluminescence signals were visualized by ECL detection kit (Millipore).

9. Transfection with p53 shRNA

A549 and H460 NSCLC cancer cells were seeded in 60-mm dishes at 5 × 10⁵ cells/dish, incubated overnight, and then transfected with p53 shRNA using Lipofectamine 3000 transfection reagent (Invitrogen, USA) according to the manufacturer’s protocol. After a 24 h transfection period,

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cells were treated with EMMQ for 48 h. Cell lysates were collected for western blot analysis.

10. Xenograft tumor evaluation

The athymic nu/nu female mice (BALB/c) of 3-4 week of age were obtained from the National Laboratory Animal Center (Tainan, Taiwan) and housed under pathogen-free conditions with a 12 h light/12 h dark schedule in the Animal Resource Facility at the animal center (Kaohsiung Medical University, Kaohsiung, Taiwan) in accordance with the

Institutional Animal Care and Use Committee guidelines. The animal study was performed according to protocols of the Institutional Animal Guidance. A total of 1×10⁶ cells were suspended in 100 μL of PBS and were injected into dorsal legs hypodermic area of nude mice with a total of 6 mice in each group. The EMMQ was dissolved in DMSO and the mixture of PBS and Matrigel™ Basement Membrane Matrix (BD Biosciences, San Jose, CA) (4:1) before EMMQ treatment. Once the tumors reached 50 mm³, the mice were injected subcutaneous with 200 μL of EMMQ (at a dosage of 1 mg/kg/mouse) or vehicle control twice a week for four consecutive weeks. The size of each tumor was measured at each time point before EMMQ administration. The tumor volumes were calculated according to the formula: 1/2 × (length×width²). After 35 days of drug treatment, mice were sacrificed by CO2 inhalation and death was confirmed by cervical dislocation. The tumor samples were dissected were measured and lysed later for protein analysis. One-way ANOVA test was used for statistical comparisons between different groups.

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11. Cell migration assay

Migration was determined using the wound healing assays by A549, H1299 and H460 cells, respectively. Cells were seeded at 2 ×10⁵ cells per well into 12-well plates for 16 h to allow cell attachment. Each NSCLC cells were treated different concentrations of EMMQ or solvent control DMSO and scraped with a 200 µL tip (time 0). Before imaging,

suspended cells were washed off. The distance of migrating cells was measured from images at 48 h after EMMQ treatment. The results of wound healing assay were normalized as ratio of wound repaired area to the non-treated control set to 100%.Wound closure was evaluated and photographed at 48 h with an inverted microscope (Nikon, Japan).

12. Gelatin zymography

A549 cells were starved for 24 h with serum-free medium.

Subsequently, cells in media containing 0.5% FBS were treated with EMMQ for different time periods and concentrations, and thereafter, the supernatants were collected. The samples were analyzed with gelatin zymography (0.1% w/v) to assess the enzymatic activity of MMPs by using gelatin as the substrate. Each lane was loaded with a total protein concentration of 3 µg and subjected to SDS-PAGE electrophoresis (30%

acrylamide, 10% SDS, 10% APS, TEMED, 1.5 M Tris (pH 8.8), 1.0 M Tris (pH 6.8)) at 48 °C. Gels were washed twice in 50 mM Tris (pH 7.4) containing 2.5% (v/v) Triton X-100 for 1 h, followed by repeated 10-min rinses in 50 mM Tris (pH 7.4). Gels were then incubated overnight in 50

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mM Tris (pH 7.5) containing 10 mM CaCl2, 0.15 M NaCl, 0.1% (v/v) Triton X-100, and 1% NaN3 at 37 °C with gentle shaking overnight. After incubation, gels were stained with 0.25% Coomassie brilliant blue and destained in 7.5% acetic acid with 20% methanol. Matrix

metalloproteinases in the loaded supernatants leads to the gelatinase bands appearing white on a blue background.

13. Statistical analysis

Experiments were performed 3 times. The differences between the treated and control cells were analyzed using the Student's t-test between two groups. The data were expressed as mean values ± SD of three independent experiments and p<0.05 was considered significant.

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V. RESULTS

1. EMMQ inhibits cell proliferation in both A549 and H460 cells To investigate the cytotoxic effect of EMMQ and to compare it with that of cisplatin in lung cancer cells, MTT assays were performed after treatment of NSCLC cells with EMMQ or cisplatin. Cisplatin reduced H460 cell growth in dose-dependent manners and cell death over 50% at 50 µM (Fig. 1B). Approximately over 100 synthetic compounds for the lung cancer screen with a known molecular weight were carried out by MTT assay. EMMQ was selected for the highest cytotoxic effect on the NSCLS cell lines. However, EMMQ induced cell death by more than 50% at 10 µM in A549 and H460 cells. The IC50 values of A549 and H460 cells were reduced with 9.7 and 9.5 μM, respectively; while no apparent growth inhibition shown in MRC-5 and H1299 cells within the concentrations studied (Fig. 1C). The results suggested that both wild type p53 NSCLC cell lines were more sensitive to EMMQ than to cisplatin.

The colony formation capacity is significantly decreased in both A549 and H460 cells by EMMQ treatment (Fig. 2A and 2B). The

numbers of colonies with in A549 and H460 cells were reduced by more than 50% when treated with 8 μM EMMQ, respectively, however there seemed no obvious inhibitory effect on null p53 H1299 cells (Fig. 2B)

2. EMMQ-induced DNA damage in wild-type p53 cell lines were examined by comet assay

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To determine whether EMMQ induced DNA damage in NSCLC cells in vitro using comet assay. The results showed that EMMQ induced DNA damage in A549 and H460 cells with wild-type p53 status. At 5 μM concentrations of EMMQ led to A549 cells a long DNA migration smear (comet tail), and these effects occurred in a dose-dependent manner (Fig.

3A), but the similar effect on H460 cells occurred at the highest dosage (10 μM). The comet tails indicating DNA lesions in A549 and H460 cells were detected after 24 h EMMQ treatment and the appearance of the excluded tail length was dose-dependent (Fig. 3B).

3. EMMQ increased sub-G1 population cells, G2/M arrest and apoptosis in both A549 and H460 cells

To gain more knowledge on cell cycle distribution following treatment, EMMQ induced cell cycle disturbance in NSCLC cells were examined. After 48 h EMMQ treatment, the cells were stained by PI and the cell cycle distribution was detected by flow cytometry. The sub-G1

cell population significantly increased in A549 and H460 cells than in DMSO vehicle controls and the effects were dose-dependent, while no effect shown in H1299 cells (Fig. 4A). In addition, our data showed that EMMQ induced A549 and H460 cells cell cycle arrest in G2/M in 8 μM at 24 h. The results indicated that EMMQ induced cell cycle arrest in G2/M at 24 h and later apoptosis at 48 h in wild type p53 A549 and H460 cells (Fig. 4B).

To examine whether EMMQ treatment induces apoptosis, NSCLC

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cells are treated with various concentrations of EMMQ (0, 5, 8, and 10 µM) and then conducted the flow cytometry-based Annexin V and PI double staining assay. Starting at a concentration of 5 μM after 48 h treatment of EMMQ, the early and late apoptotic phase cell population rose to 13% and 16% for A549 cells as well as 6% and 5% for H460 cells, respectively. However, no apoptotic cell death was shown in null p53 H1299 cells (Fig. 5A and 5B). The results implied that EMMQ-induced cell viability reduction in NSCLC cells carrying wild-type p53 was caused by apoptotic cell death following DNA damage.

4. EMMQ-induced apoptosis through intrinsic pathway

The apoptosis attributed to DNA damage can proceed through intrinsic pathway or extrinsic pathway [12, 29]. To clarify whether the intrinsic pathway is involved in EMMQ-induced apoptosis, we firstly examined the protein expression of p53, anti-apoptotic Bcl-2 family protein (Bcl-2), caspase-3 and cleavage of poly(ADP ribose) polymerase (PARP) by western bolt. Fig. 6A shows the increased concentrations of EMMQ activated p53 and down-stream effector p21, reduced p-Akt^S473, Bcl-2 and procaspase-3 levels, and increased cleavage of poly(ADP ribose) polymerase (PARP) in A549 and H460 cells after 48 h treatment.

On the other hand, in the presence of 5 μM of EMMQ, activation of p53, reduction of p-Akt^S473 and Bcl-2 intensities as well as procaspase-3 dissipation and increased PARP cleavage in A549 and H460 cells were detected in time-dependent manners. No change was shown in Akt and p-Akt^S473, Bcl-2 levels and procaspase-3 as well as cleavage PARP in

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p53-null H1299 cells within the time intervals and drug concentration ranges as studied (Fig. 6B). The results of western blots implied that EMMQ induced apoptosis through p53 activation and cleavage of PARP and diminished procaspase-3 is related to intrinsic pathway.

Mitochondria-related apoptotic pathway was linked to signal cascade following the ΔΨm disruption, signaling mitochondrial

dysfunction involvement [46]. It’s well known that the attenuated ΔΨm, outer membrane regulator Bcl-2 and release of downstream modulator mitochondrial cytochrome c indicated mitochondria-mediated pathway leading to apoptosis [47, 48]. Fig. 7A shows ΔΨm loss in A549 cells suggested that the mitochondria-mediated apoptosis by low dosage of EMMQ was initiated starting at 4 h, but the effect was absent in H1299 cells. The impaired mitochondrial functions were further accentuated by cytochrome c release in both A549 and H460 cells with increasing drug concentrations after 24 h treatment (Fig. 7B).

5. The suppressed growth of xenograft tumors in EMMQ-treated A549 cells

To assess the effect of EMMQ in vivo, a tumor xenograft study (6 animals per group) was carried out. Subcutaneous injection of EMMQ twice a week at a concentration of 1 mg/kg per mouse resulted in a

significant decrease in tumor volumes. There was also a notable decrease in the size and excised volume of tumors from EMMQ-treated mice as compared with control (Fig. 8A, 8C and 8D). The size and the excised

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volume of tumors decreased to more than 50% following 4 week treatment, while no significant difference of body weight in

EMMQ-treated nude mice relative to vehicle control as shown in Fig. 8B.

The lysates from the dissected tumors were further analyzed by western blot analysis. The levels of survival genes Akt, p-Akt^S473 and cell

proliferation marker PCNA as well as mitochondrial modulator Bcl-2 were noticeably reduced. The intensities of p53 and cleaved PARP were elevated, whereas those of procaspase-3 decreased in EMMQ-treated group as compared with those of control treatment (Fig. 8E).

6. The extent of EMMQ-induced apoptosis is dependent on p53 status To examine the role of p53 played during the process of EMMQ induced apoptosis. H1299 cells with stable expression of ectopic p53 (H1299/p53) (positive control) or mutant p53R267P (H1299/p53^R267P) (negative control) were established [49]. EMMQ induced cell viabilities significantly decrease in H1299/p53 cells as compared with

H1299/p53^R267P clone (Fig. 9A). Fig. 9B shows the loss of ΔΨm in H1299/p53 cells suggested that the mitochondria-mediated apoptosis occurred in low dosage of EMMQ, but the effect was absent in

H1299/p53^R267P clone.

Furthermore, the induced apoptotic sub-G1 cell populations by EMMQ were more apparent in H1299/p53 cells (Fig. 10). A substantial amount of cells remained at G2/M phase in H1299/p53^R267P clone.

The apoptotic cells induced by EMMQ were more distinct in H1299/p53

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than those of H1299/p53^R267P clone as determined by

annexin-V/PI-staining (Fig. 11A). The release of cytochrome c by EMMQ in H1299/p53^R267P was comparably less than that of H1299/p53 cells (Fig. 11B).

The apoptotic cells induced by EMMQ were more distinct in H1299/p53 than those of H1299/p53^R267P clone as determined by annexin-V/PI-staining (Fig. 11A). The release of cytochrome c by EMMQ in H1299/p53^R267P was comparably less than that of H1299/p53 cells (Fig. 11B). The results demonstrated that no significant effects of sub-G1 population, apoptosis ration and cytochrome c release level were detected during EMMQ concentration (0, 5, 8 and 10 μM) in

H1299/p53^R267P cells (Fig. 9, 10 and 11).

7. Down-regulated p53 abolished the onset of EMMQ-induced cell death in NSCLC cells

To ensure that p53 was indeed necessary in drug-mediated cell death, experiments by transfecting shRNA targeting exon 7 of p53 to cells prior to drug treatment were carried out along with those of NS control. The result of cell viability indicated that A549 and H460 cells were

transfected with p53 shRNA led to the sensitivity toward EMMQ was eliminated as compared with cells transfected with NS control (Fig. 13).

Western blot analysis of A549 cells showed that cells introduced with p53 shRNA exhibited significant reduction of p53 as compared with

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those transfected with NS control alone. In addition, the mitochondria modulator Bcl-2 and pro-survival gene p-Akt^S473 were unaffected by EMMQ by knocking down p53 (Fig. 14A). Similar results were also observed with reduced viabilities and Bcl-2 attenuation (Fig. 14B) in p53 shRNA-transfected H460. The results altogether suggested that p53 was needed during mitochondrial pathway activation that predates the

effectiveness of EMMQ in motivating apoptotic cell death of NSCLC cells.

8. The effect of EMMQ inhibited metastasis is dependent on Akt and β-catenin status

To gain more knowledge on cell metastasis following treatment, we examined whether EMMQ inhibited NSCLC cell migration. The

extensive evidence suggests that Akt-transduced signals directly influence cell motility in normal development and in disease [50]. These evidences

extensive evidence suggests that Akt-transduced signals directly influence cell motility in normal development and in disease [50]. These evidences