Lycorine hydrochloride inhibits cell proliferation and induces apoptosis through promoting FBXW7-MCL1 axis in gastric cancer

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: S6008-100 mL) were purchased from US Everbright®Inc. (Suzhou, China).

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 °C).

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 h. 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 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 °C incubator. LH (20 μM) was then used to treat gastric cancer cells. DMSO was applied as control. After 48 h, 10 μg/mL of BrdU was added into the medium for 2 h, and then 4% paraformaldehyde was used to fix cells for 15 min. 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.

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 h, 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 h. Finally, the results were analyzed with the Super ECL prime (US Everbright®Inc., Suzhou, China) and western blotting detection system (Qin Xiang, Shanghai, China).

After treatment with DMSO or LH at 37 °C for 48 h, 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 °C for 5 min, followed by 45 cycles of 95f for 15 s, 60 °C for 30 s and 72 °C for 90 s, then 72 °C extension for 10 min. 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.

Twenty thousand cells were seeded on the 24-well cell culture plate. After 24 h, LH (20 μM) and DMSO were added respectively, and incubated for 48 h. The PFA (4%) was used to fix the cells for 30 min. After being added PBS containing 0.5% Triton X-100 and incubated at room temperature for 5 min, the cells were added the prepared TUNEL test solution (50 μL/well) at 37 °C incubated for 60 min. 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.

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 h later. Gastric cancer cell lines MKN-45, SGC-7901 were infected with the harvested virus.

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 min, 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 1 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 2 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.

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 × 2 mm pieces and suspended in 0.1 mL 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 2 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 4 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.

We evaluated the drug combined effects on anti-tumor through Jin’s formula [34]. The Jin’s formula was as follows:

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, it was operated according to the manufacturer’s instructions.

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 h. The harvested cells were lysed by the IP lysis solution. Subsequently, it was incubated with anti-MCL1 (1%) or IgG at 4 °C 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.

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 min at room temperature. Subsequently, they were stained by DAB, and tissues were counterstained with hematoxylin. Finally, photographs were taken by the inverted microscope.

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.

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 h. 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 h, confocal microscopy was used to record the experimental results.

To investigate the effect of LH on gastric cancer cells, we treated gastric cancer cell lines, MKN-45 and SGC-7901, with different concentrations of LH (10, 20 and 40 μM) for 48 h. DMSO was used as control. The structural formula of LH was showed in Fig. S1B. MKN-45 and SGC-7901 cells exposed to LH showed that the number of cell fragments and apoptotic bodies increased in the culture medium, and the cells decreased in size and shrunk in a dose-dependent manner (Fig. S1A). Cell viability was analyzed by MTT assay and BrdU staining. MTT assay showed that LH significantly inhibited cells growth (Fig. 1a), and its semi-lethal concentration (IC50) was approximately 20 μM. In consideration of the potential toxicities of LH, we chose 20 μM LH as an indicated concentration for further investigations. BrdU staining showed that DNA synthesis decreased after treatment with 20 μM LH for 48 h (Fig. 1b). We further examined the cell cycle to assess whether LH inhibited cell proliferation by causing cell cycle arrest. Flow cytometry analysis showed that LH could arrest cell cycle progression at S phase (Fig. 1c). Furthermore, the western blotting showed that LH could significantly inhibit the expression of CDK1 and CDK2 in a dose and time dependent manner (Fig. 1d, S1C). To further investigate the effects of LH in vivo, SGC-7901 cells were injected subcutaneously into 5-week-old female nude mice. The results demonstrated that mice injected with LH had smaller tumor volume and less tumor weight comparing with the mice injected with DMSO (Fig. 1e, f). Immunohistochemical (IHC) staining with Ki67 further supported the results that LH inhibits tumourigenecity in gastric cancer cells (Fig. 1g). In conclusion, LH could dramatically inhibit the growth and tumorigenesis of gastric cancer cells.

In addition to inhibiting the proliferation of gastric cancer cells, could LH affect apoptosis or autophagy? The mRFP-GFP-LC3-adenovirus system confirmed that there was no obvious autophagy flux in gastric cancer cells after treatment with LH (Fig. S1D). By flow cytometry apoptotic analysis, we found that LH could significantly induce apoptosis of gastric cancer cells (Fig. 2a). Similarly, TUNEL staining showed that LH did induce obvious apoptosis (Fig. 2b). To further confirm these results, we tested the apoptosis-related markers, including Cleaved Caspase 3 (C-Caspase 3), Cleaved Caspase 9 (C-Caspase 9) and cleaved poly ADP-ribose polymerase (C-PARP). The western blotting results proved that C-Caspase 9, C-Caspase 3 and C-PARP increased in a concentration and time dependent manner (Fig. 2c, d).

According to the online database (https://​www.​cbligand.​org/​HTDocking/​searchstruct.​php), we observed that there might be docking sites between LH and MCL1 (Fig. S3A). As reported, MCL1 is highly expressed in gastric cancer, and the prognosis of patients with high expression of MCL1 is worse [35]. After treatment with different concentration of LH (10, 20 and 40 μM, DMSO was used as control) for 48 h, we found that MCL1 was significantly decreased in a dose dependent manner in both MKN-45 and SGC-7901 cells (Fig. 3a). Moreover, MKN-45 and SGC-7901 cells were treated with 20 μM LH for 0, 12, 24 and 48 h. The results indicated that MCL1 was reduced in a time dependent manner as well (Fig. 3a). However, BCL2 and BAK did not markedly change with the addition of LH (Fig. S3B). By using lentivirus transfection system, we obtained stable MKN-45 and SGC-7901 cell lines with exogenous overexpression MCL1. Western blotting results demonstrated that MCL1 was up-regulated after infection with lentivirus. DMSO and empty vector were used as control (Fig. 3b). The MTT experiment also proved that the growth rate of overexpression MCL1 cells after LH treatment was significantly higher than the group of empty vector treated with LH (Fig. 3c). BrdU staining experiment illustrated that overexpression of MCL1 rescued the DNA synthesis decrease induced by LH (Fig. S3E). To further explore the effect of MCL1 on cell cycle arrest induced by LH, we performed flow cytometry experiment, of which results showed that MCL1 could partly rescue the cell cycle arrest caused by LH (Fig. S3F). Subsequently, western blotting results further showed that under the LH treatment, the related cyclins (CDK1 and CDK2) were partly restored after overexpression of MCL1 (Fig. S3G).

Meanwhile, the flow cytometry for apoptotic analysis and the TUNEL staining results indicated that overexpression of MCL1 decreased percentage of apoptotic cells induced by LH (Fig. S3C, D). Besides, western blotting was performed, and C-PARP and C-Caspase 3 were partially restored in the group of MCL1 overexpression (Fig. 3d). To further explore the mechanism of apoptosis, we carried out immunoprecipitation experiment, of which results showed that the interaction of MCL1 with BIM was decreased, and the interaction of BIM with BAX was raised after treatment with different concentration gradients LH for 48 h (Fig. 3e). In conclusion, the above results showed that MCL1 could partly rescue cell proliferation and decrease apoptosis induced by LH.

To further explore the molecular mechanism of LH regulating MCL1, we performed qRT-PCR experiments. We found that the mRNA level of MCL1 did not decrease after treatment with LH, but increased slightly (Fig. 4a). Therefore, we speculated that LH might affect the protein stability of MCL1. Indeed, LH could decrease the turnover rate of MCL1 in the presence of the de novo protein synthesis inhibitor cycloheximide (CHX) (Fig. 4b). Meanwhile, the western blotting analysis revealed that the proteasome inhibitor MG132 could partly rescue the reduction of MCL1 after treatment with LH (Fig. 4c). Further, we found that LH could increase the ubiquitination levels of MCL1 (Fig. 4d). Therefore, we analyzed the related proteins which regulate the ubiquitination level of MCL1 by qRT-PCR. The primers used were listed in Table 1. The results showed that ubiquitin E3 ligase FBXW7 was up-regulated after treatment with LH (Fig. S4A). Furthermore, the western blotting showed that FBXW7 (not HUWEI or β-TrCP) was up-regulated after adding LH (Fig. 4e, S4B). After down-regulating FBXW7 in the gastric cancer cells, we found that the down-regulation of MCL1 expression induced by LH could be partially restored (Fig. 4f). Thereby, we concluded that LH could down-regulate the stability of MCL1 through FBXW7, promoting gastric cancer cells apoptosis, and inhibiting cells growth.

According to multiple lines of evidence indicated, MCL1 is commonly up-regulated in various cancers and is considered as a primary factor to resistance the treatment with the BCL2 inhibitor [36]. Besides, acquired resistance is an obstacle for most of drugs ever used in oncology. Our findings indicated that, with prolonged treatment of HA14–1, sensitive gastric cancer cell lines could spontaneously select to resistant it. So, can LH affect HA14–1-resistant cells? Based on our described screening method, we obtained drug-resistant-cell-lines (MKN-45-R, SGC-7901-R), which were resistant to HA14–1 (Fig. 5a). The IC50 of HA14–1 in BCL2-drug-resistant gastric cancer cell lines was higher than that in normal gastric cancer cell lines (Fig. S5A). Furthermore, the qRT-PCR and western blotting assays showed that the transcriptional and protein levels of MCL1 and BCL2 in BCL2-drug-resistant gastric cancer cell lines were higher than those in normal gastric cancer cell lines (Fig. S5B, C). MCL1 silencing promoted the apoptosis of drug-resistant-cell-lines, and the combination with HA14–1 (BCL2 specific inhibitor) significantly increased the apoptosis (Fig. 5b). The trypan blue staining and TUNEL staining showed that LH could also induce apoptosis in drug-resistant-cell-lines (Fig. 5c, d). Subsequently, western blotting showed that LH could also induce the up-regulation of apoptosis-related proteins, C-Caspase 9, C-Caspase 3 and C-PARP in BCL2-drug-resistant gastric cancer cell lines with a dose and time gradient effect (Fig. 5e, f). In brief, the above experiments showed that LH exactly has therapeutic effect on BCL2-resistant gastric cancer cell lines.

According to our research, the trypan blue staining showed that HA14–1 and LH could induce apoptosis in gastric cancer cell lines, respectively (Fig. 6a). Besides, the apoptosis rate induced by the combination of the two drugs was significantly higher than that sum of the ratio of apoptosis induced by the two separated drugs (Fig. 6a). Similarly, western blotting results showed that the combination of LH and HA14–1 could significantly induce the expression of apoptosis-associated proteins such as C-Caspase 9, C-Caspase 3, and C-PARP (Fig. 6b). To further investigate the treatment effects of the combination of HA14–1 and LH in vivo, we carried out the PDX model experiment. Tumor masses (GAM-AD) from The Ninth People’s Hospital of Chongqing were transplanted subcutaneously into female nude mice. After 2 weeks, the mice were randomly divided into 4 groups, then separately injected with HA14–1 (2.5 mg/kg), LH (30 mg/kg), LH (30 mg/kg) + HA14–1 (2.5 mg/kg) and DMSO once a day. Measured every 4 days, the weight of the mice did not significantly differ between the control and drugs treatment groups (Fig. 6d), indicating that the drugs were hypotoxic in mice. However, the tumor weight was significantly decreased. In addition, compared with treatment with LH only, the treatment with LH + HA14–1 have a more obvious therapeutic effect (Fig. 6c). In order to better explain the effect of drug combination, we have conducted the efficiency index (q) analysis of LH (30 mg/kg) combined with HA14–1 (2.5 mg/kg) treatment in weight of PDX tumors through Jin’s formula. The results show that LH combined with HA14–1 exhibited obvious synergistic effects (q value≥1.15), rather than the superposition effect of the two drugs (Fig. 6c). Further, the IHC results showed that the Ki67 staining of LH treated mice was significantly reduced, and the expression of Ki67 was significantly decreased after treatment with LH + HA14–1 compared with treatment with LH only (Fig. 6e). Immunohistochemical (IHC) staining with MCL1 further supported the results that LH inhibited tumorigenicity in gastric cancer through down-regulating the expression of MCL1. In a word, the results indicated that LH combined with HA14–1 exhibited a more significant inhibitory effect than LH alone in vivo.