Facile total synthesis of lysicamine and the anticancer activities of the RuII, RhIII, MnII and ZnII complexes of lysicamine

Lysicamine is a natural oxoaporphine alkaloid, which isolated from traditional Chinese medicine (TCM) herbs and has been shown to possess cytotoxicity to hepatocarcinoma cell lines. Reports on its antitumor activity are scarce because lysicamine occurs in plants at a low content. In this work, we demonstrate a facile concise total synthesis of lysicamine from simple raw materials under mild reaction conditions, and the preparation of the Ru(II), Rh(III), Mn(II) and Zn(II) complexes 1–4 of lysicamine (LY). All the compounds were fully characterized by elemental analysis, IR, ESI-MS, 1H and 13C NMR, as well as single-crystal X-ray diffraction analysis. Compared with the free ligand LY, complexes 2 and 3 exhibited superior in vitro cytotoxicity against HepG2 and NCI-H460. Mechanistic studies indicated that 2 and 3 blocked the cell cycle in the S phase by decreasing of cyclins A2/B1/D1/E1, CDK 2/6, and PCNA levels and increasing levels of p21, p27, p53 and CDC25A proteins. In addition, 2 and 3 induced cell apoptosis via both the caspase-dependent mitochondrial pathway and the death receptor pathway. in vivo study showed that 2 inhibited HepG2 tumor growth at 1/3 maximum tolerated dose (MTD) and had a better safety profile than cisplatin.


MTT assay
The cells BEL-7404, HepG2, NCI-H460, T-24 and HL-7702 were purchased from Shanghai Cell Bank in Chinese Academy of Sciences. Cells were cultured in DMEM or RPMI-1640 medium at 37 °C in a humidified atmosphere with 5% CO 2 /95% air. The stock solution of 1-4 and LY prepared as 2.0×10 −3 mol/L DMSO, and diluted to 20 M by PBS buffer when used. Cisplatin was dissolved in 0.9% sodium chloride solution and used as positive controls.
Cells were seeded in 96-well plates with 5.5 ×10 3 /180 μL per well for 24 h to reach 70% confluence, 20 μL of various concentrations of tested compounds were added to each well, each concentration was provided with 5 parallel holes. All the cells were incubated with test compounds for 48 h before 10 μL of MTT (5 mg/mL in PBS) was added to each well, and cells were incubated for another 4 h. after removed the medium, 150 μL DMSO were added to dissolve the formazan crystals, and the absorbance was recorded by enzyme labelling instrument with 490 nm/630 nm double wavelength measurement. The antitumor inhibition rate was evaluated based on the percentage of cells survival compared with the untreated cells. The IC 50 value was defined as complex concentration killing 50% cells in comparison with control cells, which calculated by the Bliss method (n = 5). All tests were repeated in at least three independent trials.

Uptake of rhodium in HepG2 cells
About 1×10 6 HepG2 cells were treated with 2 (7.0μM) and 3(14.0 μM) for 24 h, respectively. Then the treated cells were harvested and dissolved in 1 M NaOH (1 mL) and diluted with 2% (v/v) HNO 3 (5 mL) for determining the whole cell Rh(III), Mn(II) and Pt(II) content. The cells' nucleus fraction and mitochondria fraction were isolated using the FractionPREP kit from BioVision according to the instructions and digested with HNO 3 , and then diluted with double-distilled water to obtained a final solution with concentration of 5% HNO 3 (5 mL). The amount of each metal were determined by plasma-mass spectrometry (ICP-MS).

Cell cycle analysis
Cell cycle progression was determined by flow cytometric analysis. About 5×10 6 cells/well HepG2 and NCI-H460 cells were treated with 2 and 3 at various doses (3.5, 7.0, 14.0μM for 2; 7.0, 14.0 and 28.0 μM for 3) for 24 h. After incubated with 2 and 3, cells were trypsinized, collected, and fixed in ice-cold 75% ethanol overnight at -20 °C. The next day, the fixed cells were washed with icecold PBS and resuspended in 0.5 mL of PBS containing 100 μg/mL RNase, 50 μg/mL PI in the dark for 5-10 min. The cell cycle distribution was analyzed by FACS Calibur flow cytometer (BD) and calculated using ModFIT LT software (BD).

Apoptosis assay
The method of incubation HepG2 and NCI-H460 cells with 2 and 3 were the same as the cell cycle analysis assay. www.impactjournals.com/oncotarget/

Oncotarget, Supplementary Materials 2017
After treated, the cells were collected, and suspended in the annexin-binding buffer (5 × 10 5 cells/mL), then treated with annexin V-FITC and PI for 1 h at room temperature in the dark and immediately analyzed by flow cytometry.

Hoechst33258 assay
The morphology characters of apoptosis of HepG2 cells treated with 2 and 3 were carried out by Hoechst 33258 staining. About 1×10 6 cells were seeded in six-well plates, and treated with 2 (3.5, 7.0 and 14.0 μM) and 3 (7.0, 14.0 and 28.0 μM) for 24 h. The cells were fixed with carnoy-fixe solution for 10 min at room temperature and then stained with Hoechst 33258 for another 10 min in the dark. After washing three times with PBS, the HepG2 cells' morphological features of apoptosis were captured by fluorescence microscope with excitation wavelength of 330-380 nm. Apoptotic cells were defined based on the nuclear morphology changes such as chromatin condensation and fragmentation.

Caspase-3, -8 and -9 activity determinations by flow cytometry
CaspGLOW fluorescein active caspase-3/-8/-9 staining kit was used in this assay. HepG2 and NCI-H460 cells treated with IC 50 value of 2 and 3 for 24 h, and harvested at a density of 1 × 10 6 cells/mL, and then resuspended in 300 μL volume with PBS contains 1 μL of caspase-3 inhibitor (FITC-DEVD-FMK), caspase-8 inhibitor (FITC-IETD-FMK) or caspase-9 inhibitor (FITC-LEHD-FMK), respectively, and incubated for another 1.0 h at 37 °C in 5% CO 2 incubator. The cells were harvested by centrifugation, then examined used a FACSAria II flow cytometer equipped. The results were represented as the percent change on the activity comparing with the control.
Cell cycle-, apoptotic-associated proteins used western blotting analysis

Extraction of total protein
After HepG2 cells incubated with 2 (3.5, 7.0, 14.0 μM) and 3 (7.0, 14.0, 28.0 μM) for 24 h, cells were harvested and lysed used 149μL RIPA and 1μL PMSF on ice. The suspension sample was centrifuged at 12 000 rpm at 4 °C for 10 min, the supernatant liquid (total protein) was extracted, the total protein stocked in-80 °C refrigerator before used. The total protein absorbance value of the 562 nm was measured by the enzyme marker, and the protein concentration was calculated according to the standard curve [7].

Determination of cell cycle and apoptotic related proteins
The quantitative determination of protein according to the manufacturer's instructions. 25μL SDS-PAGE protein sample buffer was added to 100 μL total protein liquid and then was boiled for 5 min. 10 μL protein sample was loaded onto 10% SDS-PAGE gels and then transferred onto a PVDF membrane. The membrane was blocked with 5% BSA in TBST buffer for more than 2 h. After moving the TBST buffer, membranes incubated with an primary antibodies in TBST overnight at 4 °C and then washing with TBST three times, the membranes incubate with antimouse or anti-rabbit secondary antibodies (anti-CDK2, CDK6, Cyclin A2, Cyclin D1, Cyclin B1, PCNA, p53, pp21, p27, Apaf-1, cytochromec, caspase-3/-8/-9, c-myc, Bax, Bcl-2, PARP, and β-actin) for 1 h. The protein bands were visualized using chemiluminescence substrate.

Gene expression by a panel of genes for RT-qPCR array
The DNA chip analysis was carried out according to our previously report [3]. Differential expression profiles of cell cycle and apoptosis-related genes were analyzed using the human cell cycle PCR array(PAHS-020Z) and human apoptosis PCR array(PAHS-012Z), which implemented by Kangchen Biotech (Shanghai, China).
HepG2 cells incubated with 2 (7.0 μM) for 24 h, then 1 mL trizol was added to 15 cm 2 adherent cells, RNA was extracted according to standard protocols and converted to first strand cDNA using the RT2 First Strand Kit. Then added with RT2 SYBR Green qPCR Master Mix and each of the respective forward and reverse primers and RT-qPCR was performed. The threshold cycle (Ct) values for all the genes on each PCR Array were calculated using the instrument specific software, and the fold-changes in gene expression for pairwise comparison were calculated using the 2 −ΔCt method.

DNA binding experiment
In the DNA binding experiment, ct-DNA was stock at 4 °C with 2×10 −3 M (solute in TBS buffer) for no more than 3 days before used. Complexes and LYwere all prepared as 2×10 −3 M DMSO stock solutions, the DMSO is limited in 1% in final working solutions.

CD absorption spectrometry assay
In the CD absorption spectrometry, 150 μL2×10 −3 M ct-DNA and 2850 μL TBS were added to a cuvette, the ct-DNA CD spectrum as control. LY, 2 and 3 were added into the ct-DNA solution gradually, with the [compounds] / [DNA] ratio were 0:10, 0.5:10, 1:10, 1.5:10, 2.0:10 and 2.5:10. The working solution was incubated for 10 min after each addition and then its CD spectrum was recorded at 100 nm/min scan rate. The CD signals of the TBS were subtracted as the background.

Agarose gel electrophoresis assay
In plasmid DNA unwinding experiments, 1 μL 0.5 μg/μL supercoiled pBR322 DNA was treated with different concentration of LY, 2 and 3 in TBE buffer at 37 °C in the dark for 4 h. Then each sample mixed with loading buffer in 5:1(v/v), 12μL sample analyzed by 1% agarose gel electrophoresis at 5 V/cm in 1×TBE buffer solution. Finally, the gel was then stained by EB (0.5 μg/mL) for 20 min and photographed via a BIO-RAD imaging system under a UV-Vis transilluminator.

In vivo tumour growth inhibition experiment Animal used
Animals were supplied by Guangxi Medical University Laboratory Animal Centre (Guangxi, China, approval no. SCXK 2014-0002 andSYXK 2014-0003), and the animal experiment were carried out at there. KM mice, half male and female, 20-23g, 5-6 weeks old for acute toxicity test. BALB/c nude mice, male, 19-21 g, 5-6 weeks old for antitumor xenograft experiment. Animals were housed at a sterile environment with conditions of constant photoperiod (12 h light/12 h dark at 25-28 °C and 45%-65% humidity), in addition, BALB/c nude mice housed in individual ventilated caging system (IVC Rack).

Maximumt tolerated dose (MTD)
The MTD was determined using male and female KM mice. The large concentration of 2 in 10% (v/v) DMSO solution (0.57mg/mL) was single intraperitoneal injected to 10 KM mince with 0.4 mL/10 g (i.e., 22.8 mg/ kg). The body weight of mice was evaluated daily for the first 5 days and then twice a week thereafter. The MTD was defined as the largest administered dose of drug and route cause a mean body weight loss≤20%, no animal death and the reversible and temporary toxicities [8].