Reagents and antibodies
The MCL1 (16225-1-AP), HA (51064-2-AP), Alpha Tubulin (11224-1-AP) and FBXW7 (28424-1-AP) antibodies were purchased from Proteintech Group (Wuhan, China). The CDK1 (#77055), CDK2 (#78B2), Cleaved Caspase 3 (#14220), Cleaved Caspase 9 (#52873), Cleaved PARP (#5625), PARP (#9542), Bim (#2933), BAX (#5023) and BCL2 (#15071) antibodies were purchased from Cell Signaling Technology (CST, Boston, MA, USA). Cycloheximide (C7698), 5-Bromo-2-deoxyuridine (BrdU), 3-(4, 5-Dimethylthiahiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, M5655), dimethyl sulfoxide (DMSO, D5879) and Z-Leu-leu-leu-al (MG132, M7449) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-BrdU (ab8152) and Ki67 (ab15580), β-TrCP (ab71753) and HUWEI/mule (ab70161) antibodies were purchased from Abcam (Cam-bridge, MA, USA). The One Step TUNEL Apoptosis Assay Kit (C1089), RIPA lysis buffer, Phenylmethanesulfonyl fluoride (PMSF) and BCA protein assay kit were purchased from Beyotime (Shanghai, China). Lycorine hydrochloride was purchased from MUST BIO-TECHNOLOGY (Cheng Du, China). Alexa Fluor 488 goat anti-rabbit IgG (H+L) (35552) was purchased from Invritrogen (California, USA). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), puromycin (A1113803) were purchased from Life Technologies (New York, USA). The transfection reagent Lipofectamine™ 2000 was obtained from Thermo Fisher Scientific (New York, USA). HRP goat anti-mouse and goat anti-rabbit antibodies were purchased from Beyotime (Shanghai, China). Annexin V-APC (2005128) and propidium iodide (2048964) were obtained from Invitrogen (California, USA). Super ECL prime (Cat: S6008-100mL) were purchased from US Everbright®Inc. (Suzhou, China).
Cell Culture
MKN-45, SGC-7901 and 293-FT cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All the cell lines were free of mycoplasma contamination as tested by vendors using MycoAlert kit from Lonza. MKN-45-R and SGC-7901-R cell lines were obtained from our laboratory. MKN-45, SGC-7901, MKN-45-R and SGC-7901-R cell lines were cultured in 1640 medium with 1% Penicillin-Streptomycin Solution and 10% fetal bovine serum (Biological Industries, BI, Beit HaEmek, Israel). 293-FT cells were cultured in DMEM medium with 2% glutamine (Biological Industries, Connecticut, USA), 1% non-essential amino acids (Biological Industries, Connecticut, USA), 1% sodium pyruvate (Biological Industries, Connecticut, USA), and 10% fetal bovine serum (BI). The above cell lines were cultured in standard conditions (5% CO2, 37 ℃).
Cell viability detection.
MTT assay was performed to detect cell viability as previously described [30]. Simply put, SGC-7901 and MKN-45 cells in logarithmic phase were counted and seeded in 96 well plates (800 cells in 200 μL medium per well) and then attached overnight. RPMI-1640 complete medium with different concentrations LH (10, 20 and 40 μM) were used to treat SGC-7901 and MKN-45 cells, respectively. DMSO was used as control. MTT (5 mg/mL, 20 μL per well) was added into the culture medium at the designated time points and cultured at 37°C incubator for 2 hours. Formazan was further dissolved with DMSO (200 μL). The absorbance at 560 nm was monitored by the microplate reader (Thermo Fisher, Waltham, Ma, USA). All the experiments were carried out independently in triplicates. The data were analyzed by the Graphpad.
BrdU staining
BrdU staining was used to monitor the cell proliferation as previously described method [20]. 2×104 SGC-7901 or MKN-45 cells in logarithmic phase were inoculated into 24 well plates and cultured overnight in 37℃ incubator. LH (20 μM) was then used to treat gastric cancer cells. DMSO was applied as control. After 48 hours, 10 μg/mL of BrdU was added into the medium for 2 hours, and then 4% paraformaldehyde was used to fix cells for 15 minutes. After treatment with 2 M HCL and 0.3% Triton X-100, the cells were blocked with 10% goat serum (zsgb bio, Beijing, China). The cells were then successively incubated with BrdU primary antibody and then with secondary antibody. Before microscopic observation, the cells were stained with DAPI. BrdU positive cells were counted in three random areas.
Western blotting assay
Cells were collected and then lysed in a RIPA lysis buffer with phenylmethanesulfonyl fluoride (PMSF) as previously described [31]. Protein concentration was detected with a BCA protein assay kit. The protein was separated on 8%-12% SDS-PAGE Gels, and then transferred to a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% BSA at room temperature for 2 hours, the PVDF membrane was incubated with a diluted primary antibody overnight at 4°C. The next day, the PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibody (HRP-conjugated secondary antibodies) at room temperature for 2 hours. Finally, the results were analyzed with the Super ECL prime (US Everbright®Inc., Suzhou, China) and western blotting detection system (Qin Xiang, Shanghai, China).
Quantitative and reverse transcriptional PCR
After treatment with DMSO or LH at 37 °C for 48 hours, cells were harvested and total RNA was isolated from cells using Trizol reagent according to the manufacturer's instructions as previously described [32]. Total RNA was reverse-transcribed to cDNA using M-MLV reverse transcriptase (Promega, Wisconsin, USA). The qRT-PCR was performed in 20 μL reaction mixture, containing 2 μL cDNA template, 10 μL 2×GoTaq® qPCR Master Mix (Promega, USA), 0.5 μL forward primers (Huada Gene, China), 0.5 μL reverse primers (Huada Gene, China) and 7 μL nuclease-free water. The amplification program went as follows: 95℃ for 5 minutes, followed by 45 cycles of 95f for 15 seconds, 60℃ for 30 seconds and 72℃ for 90 seconds, then 72℃ extension for 10 minutes. The normalized expression control was based on the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) value. Then, the relative mRNA expression levels were quantified by using the 2-∆∆Ct method. All primers were showed in Table 1. All the qRT-PCR results were repeated three times.
Flow cytometry assay
(a) Cell cycle analysis: MKN-45 and SGC-7901 cells were harvested after treatment with LH (20 μM) for 48 hours, and fixed in 70% ethanol at 4°C for 24 hours. After being washed with PBS, the cells were added 2 μL RNaseA (4mg/mL) and 5 μL propidium iodide for 35 minutes at 37 ℃. Subsequently, the BD Accuri C6 flow cytometer and FlowJo software were used to analyze the change of DNA.
(b) Cell apoptosis analysis: MKN-45 and SGC-7901 cells were harvested after treatment with LH for 48 hours. After being washed with 1×Bingding buffer, the cells were incubated with Annexin V-APC (5 μL) and propidium iodide (5 μL) at room temperature for 20 minutes. Subsequently, the BD Accuri C6 flow cytometer and FlowJo software were used to analyze the change of apoptosis following the manufacturer’s instructions.
TUNEL experimental analysis
Twenty thousand cells were seeded on the 24-well cell culture plate. After 24 hours, LH (20 μM) and DMSO were added respectively, and incubated for 48 hours. The PFA (4%) was used to fix the cells for 30 minutes. After being added PBS containing 0.5% Triton X-100 and incubated at room temperature for 5 minutes, the cells were added the prepared TUNEL test solution (50 μL/well) at 37 ℃ incubated for 60 minutes. Finally, the result was observed by fluorescence microscope after sealing with anti-fluorescence quenching solution. The excitation wavelength of Cy3 is 550 nm, and the emission wavelength is 570 nm.
Transfection and infection
The plasmid shMCL1 (#1, #2), shFBXW7 and the negative control (SHC002) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The MCL1-overexpression plasmid (PCDH-CMV-MCS-EF1-copGFP-MCL1) was purchased from YouBio (Changsha, China) [19]. Transfection and infection were carried out as manufacturer’s instructions. Firstly, the liposomes and the packaging plasmids (PLP1, PLP2, VSVG, target plasmids: shMCL1, shFBXW7 or OE-MCL1) were transferred into 293-FT cells. The virus was harvested 48 hours later. Gastric cancer cell lines MKN-45, SGC-7901 were infected with the harvested virus.
Subcutaneous tumor xenografts (CDX model)
Experiments in vivo were carried out with the approval of the Committee for Animal Protection and Utilization of Southwest University. According to the Guidelines for Animal Health and Use (Ministry of Science and Technology, China, 2006), all experiments were conducted orderly. Purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China), six five-week-old female nude mice were raised and observed in SPF room for a week to adapt to the new environment. On June 20, 2019, SGC-7901 cells (1×106 cells per mouse) suspended in 0.1 mL serum-free RPMI-1640 were subcutaneously inoculated on the left and right upper back of mice. In order to alleviate the pain of mice, we used the isoflurane for nasal anaesthesia before subcutaneous injections. Isoflurane can make the mice enter anesthesia state faster and recover quickly. After the anesthesia stops, the mice commonly wake up within 2 minutes, and the control of anesthesia depth was very easy. If the mice were found to be out of condition during the operation, the anaesthesia machine would be shut off immediately, and the mice would be rescued quickly. Hence, the safety of mice was guaranteed. Isoflurane can be completely discharged from the alveoli through respiration without affecting metabolism in vivo, and has no effect on the experimental results. In addition, isoflurane is widely used in animal experiments in the world. The mouse anaesthesia system was purchased from Reyward Life Technology Co., Ltd. (Shenzhen, China). All experiments were performed on a sterile workbench of an SPF room [33]. Before and after subcutaneous injection, 75% medical alcohol was used to disinfect the epidermis of mice. After one week injection, they were randomly divided into two groups, which were respectively treated with DMSO and LH (30 mg/kg) once a day for 16 days. During this period, tumor volume [tumor volume= (length×width2)/2] was measured every two days under strict and standardized feeding conditions. Before tumor collection, nasal anesthesia (isoflurane) was used in mice to relieve pain. Then, the mice were killed by cervical dislocation. The tumor was removed, and the weight was recorded. The bodies were frozen at -20°C before transferring to Laibite Biotech Inc. (Chongqing, China) for incineration. Finally, the tumor was photographed and recorded, which will be used for subsequent immunohistochemistry experiments.
PDX experiment
Purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China), 20 five-week-old female nude mice were raised and observed in SPF room for a week to adapt to the new environment. On August 5, 2020, with the consent of the patient's family, the GAM-AD tumor mass from The Ninth People’s Hospital of Chongqing was cut into even 2×2×2mm pieces and suspended in 0.1mL serum-free RPMI-1640, and planted into subcutaneous tissue of mice in equal volume. The whole process was consistent with the previous subcutaneous tumor xenografts experiment. After two weeks, they were randomly divided into four groups, respectively treated with DMSO, LH (30 mg/kg) and LH (30 mg/kg)+HA14-1 (2.5 mg/kg) once a day for 13 days. In addition, the weight of the mice was recorded every four days. The process of tumor collection was also consistent with the aforementioned subcutaneous tumor xenografts experiment. The tumor weight was recorded, and the tumor was photographed and recorded.
Jin’s formula
We evaluated the drug combined effects on anti-tumor through Jin’s formula [34]. The Jin’s formula was as follows:
Immunoprecipitation (Co-IP)
Protein A/G Magnetic Beads (HY-K0202) were purchased from MCE (Monmouth Junction, NJ, USA). Cells treated with LH or DMSO were lysed and collected. Then, operate according to the manufacturer's instructions.
Ubiquitination assay
Firstly, the HA-Ub plasmid was transiently transferred into 293-FT. Then 293-FT cells were harvested before incubating with proteasome inhibitor MG132 (50 μg/mL) from Selleck (Houston, USA) for 6 hours. The harvested cells were lysed by the IP lysis solution. Subsequently, it was incubated with anti-MCL1 (1%) or IgG at 4 ℃ for overnight. The second day, the Protein A/G Magnetic Beads were added following with the instruction. Then, the proteins adsorbed from magnetic beads were analyzed by western blotting. HA tag antibody (51064-2-AP) from Proteintech Group (Wuhan, China) was used to check the interaction between MCL1 and Ub.
Immunohistochemistry Assay
After the tumor tissues were paraffin sectioned, they were incubated with MCL1 (1:100) FBXW7 (1:100) or Ki67 (1:100) antibodies at 4°C for overnight, then incubated with HRP-conjugated secondary antibodies for 20 minutes at room temperature. Subsequently, they were stained by DAB, and tissues were counterstained with hematoxylin. Finally, photographs were taken by the inverted microscope.
The screen of BCL2-drug-resistant-cell-lines
The MKN-45 and SGC-7901 cell lines were cultured in 1640 complete medium with HA14-1(9 μM) for a week. Then the dead cells were washed with PBS. The remaining living cells were diluted in 96-well plate by gradient dilution method and maintained a high concentration of HA14-1. Continue to cultivate for 3 weeks. Finally, the surviving monoclonal cells were selected, and amplification cultured. Finally, the HA14-1-resistant cell lines (MKN-45-R and SGC-901-R) were obtained.
Autophagy flux detection
The mRFP-GFP-LC3-adenovirus (HB-AP210 0001) was purchased from HanBio (Shanghai, China). Subsequently, mRFP-GFP-LC3B-adenovirus was added into the medium of MKN-45 and SGC-7901, cultured for 24 hours. Then, the old medium was removed and fresh 1640 medium (with 10 % fetal bovine serum, 1% Penicillin, Streptomycin, and 20 μM LH) was added. After 48 hours, confocal microscopy was used to record the experimental results.
Statistical analysis
Statistical analyses were obtained by the Graphpad prism software. All observations were confirmed by at least three independent experiments. Data were showed as MEAN±SD and analyzed by unpaired 2-tailed t-test. P-values of <0.05 (*), <0.01 (**), and <0.001 (***) were considered statistically significant.