br Fig a novel indolo j phenanthridine alkaloid isolated
(Fig. 1), a novel indolo [3,2-j] phenanthridine alkaloid, isolated from cyanobacteria Calothrix in 1999 and was reported as a weak Top I in-hibitor . CAA showed potent anti-proliferative activities against a series of cancer cell lines including Hela cells, cultured (p53 proficient) CEM Leukemia cells and Jurkat cells, with IC50 values of 40, 200 and 1600 nM, respectively [5–7]. CAA was also reported to induce cell apoptosis in Jurkat cells. However, the exact mechanism how CAA in-duces cancer cell apoptosis remains unclear.
Our laboratory has been searching for novel anti-cancer agents [8–12], and we have completed the total synthesis of CAA . A series of CAA analogues were synthesized and screened for their anticancer activities. Some of them displayed anti-cancer eﬀects at nanomolar concentrations (data not shown). CAA45 (Fig. 1) is a CAA analogue with excellent anti-proliferative eﬀect, especially against lung cancer cells. In the present study, we planned to get further insight on the anti-lung cancer activity of CAA45 and its potential eﬀect mechanism.
O NH Cl
2. Materials and methods
2.1. Chemicals and materials
CAA and CAA45 were synthesized in our lab . Camptothecin (CPT) was obtained from Shanghai Macklin Biochemical Co. Ltd. and doxorubicin hydrochloride (DOX) was purchased from Shanghai Sangon Biotech Co. Ltd. Compounds were dissolved in DMSO at 10 mM as a stock solution, and diluted with culture medium. The maximum concentration of DMSO used in biological assays is < 0.1% (v/v) in the medium. For the in vivo drug administration, compound CAA45 was dissolved in sterilized ultra-pure water at 2 mg/mL, and the positive control drug CPT was dissolved in intralipid 20% at 1 mg/mL, the ve-hicle group was intralipid 20% without any drug. All the stock solutions were stored at −80 °C prior to use.
Cleaved caspase-3 (C-caspase-3), cleaved caspase-8 (C-caspase-8), cleaved caspase-9 (C-caspase-9), Bad, Bax, Bcl-2, Bcl-xl, phospho-Akt (p-Akt), phospho-JNK (p-JNK), p53 and LC3B NB-598 were pur-chased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Cytochrome C (Cyto-C), Cox IV, Akt, JNK, MMP-2 and MMP-9 were purchased from Abcam Biotechnology (Cambridge, MA, USA). The β-actin antibody and goat-anti-rabbit horseradish-peroxidase-conjugated (HRP) secondary antibodies were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China).
Two NSCLC cell lines A549 and NCI-H1650, and two human normal cell lines BEAS-2B and L02 (human bronchial epithelial cell line BEAS-2B; human liver cell line L-02) were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). A549 and BEAS-2B cells were maintained in DMEM (HyClone) medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. NCI-H1650 and L-02 cells were cultured in RPMI-1640 (HyClone) medium supplemented with 10% FBS, 1% penicillin and streptomycin. All the cells were cultured at 37 °C in a 5% CO2 humidified atmosphere.
2.3. Cell viability assay
Cell viability upon treatment of the compounds was determined by using Cell Counting Kit-8 assay (Sangon Biotech, Shanghai, China) following the manufacturer's instructions. Cells were seeded in 96-well plates at density of 5 × 103 per well and were incubated for 72 h with either vehicle (DMSO) or desired concentrations of compounds at 37 °C in a 5% CO2 incubator. Then, cells were incubated with CCK-8 solution for 1 h at 37 °C in a 5% CO2 incubator. Finally, the absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Shanghai, USA). Cell viability was calculated as the ratio of the ab-sorbance of the treated cells to the absorbance of the control groups. IC50 values were calculated by GraphPad Prism 6, which were the mean values derived from three independent experiments. Life Sciences 219 (2019) 20–30
2.4. Colony formation assay
Colony formation assay was used to evaluate the anti-proliferative eﬀect of CAA45 against A549 and NCI-H1650 cells as previously re-ported . Varying cell numbers were seeded in 6-well plates and incubated at 37 °C for 24 h. Then the cells were treated with either DMSO or diﬀerent concentrations of CAA45 (0.01, 0.03 and 0.06 μM) for 24 h, and then replaced by compound-free media for every 3 d. After 14 d cell plating, cell colonies were fixed with 4% paraformaldehyde for 15 min, and stained by 0.1% crystal violet for 30 min at room tem-perature. Colonies with over 100 cells were defined as positive.
2.5. DNA Top I inhibition assay
The inhibition of CAA45 on DNA Top I was performed indirectly by evaluating the relaxation of supercoiled pBR322 plasmid DNA. The experimental conditions were as previously reported . Specifically, each reaction mixtures contained 2 μL 10× DNA Topo I buﬀer, 2 μL 0.1% BSA, 0.5 U Topo I protein, 0.25 μg of pBR322 plasmid DNA (Ta-kara, Japan) and the test compound CAA45 (1, 5, and 10 μM) or posi-tive control CPT (while the drug was omitted in control panel (panel 2) representing the capability of Topo I activity), then deionized water was used to complement the reaction volume to 20 μL. The reaction was performed at 37 °C in a water bath for 30 min and stopped by adding 1 μL 10% SDS and 3.5 μL 6× loading buﬀer. Finally, the reaction mixtures were separated by electrophoresis on a 1% agarose gel at 100 V for 40 min. The gels were stained with 0.5 mg/mL ethidium bromide for 15 min and visualized by using Molecular Imager FX (Biorad, Hemel Hempsted, UK).