Antiproliferative effect of a novel synthesized carbazole compound on A549 lung cancer cell line.

M. Med. Sc. University of KwaZulu-Natal, Durban 2014.Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3-en-2-one (ECAP) to inhibit the proliferation of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipid peroxidation and comet assays were used to assess the anti-proliferative effects of the compound on A549 lung cancer cell line. Protein expression was determined using western blots, apoptosis was measured by luminometry for caspase-3/7, -8 and -9 and flow cytometry was used to measure phosphatidylserine externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells by significantly down-regulating the expression of antioxidant defense proteins, Hsp70 (p < 0.02) and Bcl-2 (p < 0.0006), thereby up-regulating reactive oxygen species (ROS) production. This resulted in DNA damage (p < 0.0001) and subsequent up-regulation of Bax and caspase activity consequently inducing apoptosis of lung cancer cells. These results demonstrate the potential anticancer effects of ECAP on cultured lung cancer cells. However, further investigation and characterization is required to fully understand the possible use of carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3- en-2-one as potential lung cancer treatment

Cytotoxic effect of a novel synthesized carbazole compound on A549 lung cancer cell line

Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3-en-2-one (ECAP) to induce cytotoxicity of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipid peroxidation and comet assays were used to assess the cytotoxic effect of the compound on A549 lung cancer cells. Protein expression was determined using western blots, apoptosis was measured by luminometry (caspase-3/7, -8 and -9) assay and flow cytometry was used to measure phosphatidylserine (PS) externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells due to a significant reduction in the expression of antioxidant defence proteins (Nrf2 and SOD), Hsp70 (p < 0.02) and Bcl-2 (p < 0.0006), thereby up-regulating reactive oxygen species (ROS) production. This resulted in DNA damage (p < 0.0001), upregulation of Bax expression and caspase activity and induction of apoptosis in lung cancer cells. The results show the anticancer potential of ECAP on lung cancer.S1 Fig. MTT assay measured in untreated (control) and 1% DMSO (vehicle control) treated A549 cells. The data showed no significant cytotoxicity.S2 Fig.Western blots showing the effect of ECAP on the expression of Bax, Bcl-2, p53, Nrf2, Hsp70 and SOD. The original western blots which were used for western blot analysis (Fig 6).College of Health Sciences, University of KwaZulu-Natalhttp://www.plosone.orgam201

Cytotoxic Effect of a Novel Synthesized Carbazole Compound on A549 Lung Cancer Cell Line

<div><p>Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3-en-2-one (ECAP) to induce cytotoxicity of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipid peroxidation and comet assays were used to assess the cytotoxic effect of the compound on A549 lung cancer cells. Protein expression was determined using western blots, apoptosis was measured by luminometry (caspase-3/7, -8 and -9) assay and flow cytometry was used to measure phosphatidylserine (PS) externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells due to a significant reduction in the expression of antioxidant defence proteins (Nrf2 and SOD), Hsp70 (p < 0.02) and Bcl-2 (p < 0.0006), thereby up-regulating reactive oxygen species (ROS) production. This resulted in DNA damage (p < 0.0001), up-regulation of Bax expression and caspase activity and induction of apoptosis in lung cancer cells. The results show the anticancer potential of ECAP on lung cancer.</p></div

Time dependent effect of ECAP on caspase activity and ATP level.

<p>(A) The activity of Caspase-3/7 (6 h: p < 0.4036, 24 h: p < 0.0003), (B) Caspase-8 (6 h: p < 0.4364, 24 h: p < 0.0001), (C) Caspase-9 (6 h: 0.4171, 24 h: p < 0.0124) and (D) ATP (6 h: 0.4011, 24 h: 0.0011) levels in A549 lung cancer cell line after 6 h and 24 h incubation. [* denotes statistical significance with respect to the control and uncertainties represent standard deviation (SD) from the means. Number of replicates: n ≥ 3].</p

The effect of ECAP on the induction of apoptosis of A549 cells after 24 h treatment.

<p>[(p < 0.0001), *** significance compared to the control and number of replicates: n ≥ 3].</p

Characterization of the novel (Z)-4-((9-ethyl-9H-carbazol-3-yl) amino) pent-3-en-2-one (ECAP).

<p>(A) IR, (B) ¹H-NMR and (C) ¹³C-NMR spectrums.</p

Schematic preparation of carbazole ECAP in Cl₃/Ethanol, reflux, 5 h RT.

<p>Schematic preparation of carbazole ECAP in Cl₃/Ethanol, reflux, 5 h RT.</p

Comet assay showing DNA damage in A549 cells after 24 h treatment.

<p>(A) Control and (B) ECAP treated A549 cells. (p < 0.0001).</p

Western blot data shwing effect of the novel ECAP on protein expression in A549 lung cancer cell line.

<p>(A) Bax, (B) Bcl-2, (C) p53, (D) Nrf2, (E) Hsp70 and (F) SOD.</p

Dose-response curve showing the cytotoxic effect of ECAP and ellipticine on PBMCs and A549 lung cancer cell line.

<p>(A) The effect of (A) ECAP and (B) ellipticine on cell viability of PBMCs and A549 cells after 24 h treatment. [N = 3 replicates at 95% confidence interval].</p