Human cervical cancer Caski, C33A, HeLa and SiHa cells were purchased from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cells were cultured at 37°C with 5% CO₂. DMEM, FBS and 1% penicillin/streptomycin were purchased from Biological Industries.

Isoliensinine (98% by high performance liquid chromatography; Beijing Solarbio Science & Technology Co., Ltd.) was dissolved in DMSO to 20 mg/ml. 0.1% DMSO was used as the vehicle control for isoliensinine.

AKT inhibitor AKTi-1/2 (10 mM; cat. no. SF2784) was purchased from Beyotime Institute of Biotechnology. The Cell Counting Kit-8 (CCK-8) was purchased from Shanghai Yeasen Biotechnology Co., Ltd. The Annexin V apoptosis kit (cat. no. LHK601-020) was purchased from Beijing Jiamei Nuno Biotechnology Co., Ltd.

Primary rabbit anti-human AKT (cat. no. 4691), phosphorylated (p-)AKT (S473) (cat. no. 4060), p21 (cat. no. 2947), CDK2 (cat. no. 2546), Cyclin E1 (cat. no. 20808), GSK3α (cat. no. 4337), Mcl-1 (cat. no. 94296), cleaved caspase-9 (cat. no. 9508), and GAPDH (cat. no. 5174) antibodies and secondary antibodies were purchased from Cell Signaling Technology, Inc. Secondary antibodies included the HRP-conjugated anti-rabbit antibody (cat. no. 7074) and HRP-conjugated anti-mouse antibody (cat. no. 7076). All primary antibodies were diluted at a ratio of 1:1,000, whilst all secondary antibodies were diluted at a ratio of 1:2,000 for use in the study.

Human cervical cancer Siha, HeLa, Caski and C33A cells were seeded into 96-well plates at a density of 8×10³ cells/well 1 day before isoliensinine treatment. After the cells were treated with 0, 5, 10, 15, 20 or 25 µM isoliensinine for 24 and 48 h at 37°C, DMEM (100 µl/well) containing 10 µl CCK-8 was added. The cells were then cultured at 37° for a further 2 h before absorbance was measured in each well at 450 nm. The cell viability was calculated by the formula [experimental optical density (OD) values-background OD values]/(control OD values-background OD values) ×100. All experiments were performed in triplicate. The IC₅₀ value was calculated by non-linear regression (curve fit) using the GraphPad Prism 8 software (GraphPad Software, Inc.). The XY drawing was first elected to create a new file and before the corresponding data were entered. The data were then converted into logarithmic format, following which ‘Transform’ and ‘Transform X values using X=Log (X)’ were selected under the ‘analysis column’. Finally, ‘non-linear expression (curve fit)’ and ‘dose-response-inhibition’ were selected for the IC₅₀ calculations.

Cervical cancer HeLa and C33A cells were plated into six-well plates at a density of 1×10³ cells/well and treated with isoliensinine at concentrations of 0, 1, 2, 4 or 8 µM at 37°C, and 0.1% DMSO was used as a negative control. The media was changed after 48 h of incubation. At 10–14 days later, when colonies were present, the cervical cancer cells were fixed with 4% methanol for 20 min and stained with 0.1% gentian violet for 30 min at room temperature. Valid clones that contained >50 cells were manually counted.

Human cervical cancer Siha, HeLa, Caski and C33A cells were seeded in six-well culture plates at a density of 5×10⁵ cells/well and then treated with isoliensinine (0–40 µM) and AKTi-1/2 (7.5 µM). After treatment with isoliensinine for 24 h at 37°C, the cells were collected in pre-cooled microcentrifuge tubes and centrifuged at 2,000 × g for 5 min at room temperature and washed once with PBS. The cells were stored at 4°C overnight after fixation with 70% ethanol. After 70% ethanol fixation, the cells were collected by centrifugation again at 2,000 x g for 5 min at room temperature, washed twice with PBS and treated with 2 µl RNase and 2 µl propidium iodide (PI). The cells were incubated at room temperature for 30 min. A total of 3×10⁴ cells were collected from each sample for flow cytometry analysis using BD Accuri™ C6 software (version 1.0.264.21; BD Biosciences).

Human cervical cancer Siha, HeLa, Caski and C33A cells were seeded into six-well culture plates at a density of 1×10⁵ cells/well. After treatment with isoliensinine (0–40 µM) and AKTi-1/2 (7.5 µM) for 48 h at 37°C, the cells were dislodged and centrifuged at 2,000 × g for 5 min at room temperature, washed once with PBS and labeled with FITC isomer for 15 min at room temperature in the dark. Prior to flow cytometry analysis, the cells were stained with PI for 5 min and mixed at room temperature in the dark. A total of 5×10⁴ cells were obtained for each sample and analyzed by flow cytometry.

During analysis, in the apoptosis diagram quadrant Q3 represents the population of early apoptotic cells whereas quadrant Q2 represents the population of late apoptotic cells. Together, these quadrants were used for quantification of apoptosis in each treatment group.

Human cervical cancer Siha, HeLa, Caski and C33A cells were incubated with 0, 5, 10, 20 or 40 µM isoliensinine for 24 h at 37°C. Subsequently, they were dislodged and collected in pre-cooled microcentrifuge tubes at 2,000 × g for 5 min at room temperature and dissolved in TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNAs were extracted and reverse transcribed into cDNAs using the RevertAid First Strand cDNA Synthesis Kit (cat. no. K1622; Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized using the temperature protocol of 65°C for 5 min, 60 min at 42°C and termination by heating at 70°C for 5 min.

Subsequent PCR was prepared using 2X Taq Master Mix (cat. no. E005-02A; Novoprotein; Jinan Protein Technology Co., Ltd.) and ran in a ProFlex PCR System (Thermo Fisher Scientific, Inc.) using the following thermocycling protocol: 94°C for 1 min for preheating, followed by 20–30 cycles at 94°C for 30 sec for denaturation, 60°C for 30 sec for annealing and 68°C for 40 sec for extension, and then a final cumulative amplification at 68°C for 10 min. GAPDH was used as the internal reference and was run for 20 cycles, whereas the target genes shown in Table I and p21, Cyclin E and CDK2 were run for 25–30 cycles. The PCR products were separated by 1% agarose gel stained with 7% GelRed (cat. no. 41003; Biotium, Inc.) and Tanon 2500 Gel Imaging System (Tanon Science and Technology Co., Ltd.) was used for imaging the gels.

For RT-qPCR, the qPCR reaction was prepared using FastStart™ Universal SYBR^® Green Master Mix (Roche Diagnostics) and analyzed using an Applied Biosystems QuantStudio 3 Real-time PCR machine with the following thermocycling reactions: 1 cycle at 95°C for 60 sec; followed by 45 cycles of 95°C for 10 sec, 60°C for 10 sec and 72°C for 10 sec; then 1 cycle at 95°C for 10 sec, 65°C for 60 sec and 97°C for 1 sec. GAPDH was used as the control to evaluate mRNA expression in each sample and mRNA expression was analyzed using the 2^−∆∆Cq method (19). The primers of CDK2, cyclin E, CDK4, Cyclin D1, p27, p15 and PTEN were designed according to previous studies (20–24) and are listed as Table I.

Human cervical cancer Siha, HeLa, Caski and C33A cells were seeded into 12-well culture plates at 1×10⁵ cells/well and were cultured overnight at 37°C. After adding 0, 5, 10, 20 or 40 µM isoliensinine, AKTi-1/2 (7.5 µM) and 0.1% DMSO as control treatment for 24 h at 37°C, the cells were washed once with PBS and dissolved in Cell Lysis Buffer for Western and IP (cat. no. P0013; Beyotime Institute of Biotechnology) supplemented with phosphatase and protease inhibitor cocktails. The cell lysates were then transferred to pre-cooled microcentrifuge tubes and centrifuged at 12,000 × g for 10 min at 4°C.

The cells were dissolved in lysis buffer (Beyotime Institute of Biotechnology) according to the number of cells, then heated at 100°C for 5 min and separated on a 10% gel using SDS-PAGE. Subsequently, the proteins were transferred onto PVDF membranes. The membranes were washed once for 10 min in PBS-T (PBS; 1% Tween-20) and blocked with 3% non-fat milk dissolved in PBS-T for 1 h at room temperature. The membranes were then incubated with the primary antibodies overnight at 4°C, washed three times with PBS-T and incubated with secondary antibodies for 1.5 h at room temperature. Finally, the membranes were washed three times with PBS-T and visualized by Western Bright™ ECL HRP substrate (Advansta, Inc.).

The complex models of human AKT1 (PDB ID, 6HHF) and AKT2 (PDB ID, 3D0E) were retrieved from https://www.rcsb.org/. Ligands data were shown as https://www.rcsb.org/structure/6HHF and https://www.rcsb.org/structure/3D0E. Ligands from AKT1/2 were separated following the prompt command using the AutoDock Tools software (http://mgltools.scripps.edu/downloads; version 1.5.6).

AKT1/2 docking was performed using the AutoDock 4.0 (http://autodock.scripps.edu/; version 4.0) and AutoDock Tools 1.5.6 software, whereas ligands (isoliensinine and AKTi-1/2) were drawn using the Chem3D (https://www.chemdraw.com.cn/; version 20.0) software. The selection of flexible residues (from docking center, AKT1: CYS296, AKT2: MET229) was based on previous reports (25,26). The docking center and grid box of AKT1was x=8.717, y=4.212 and z=11.536, and x=60, y=60 and z=66, respectively. The docking center and grid box of AKT2 was x=16.477, y=−34.368 and z=1.722, and x=60, y=60 and z=66. Independent docking calculations for AKT1/2 ligands were conducted with 250,000 evaluations using the Lamarckian genetic algorithm (27). The results were analyzed using the PyMol (https://pymol.org/2/; version 2.0) software.

Student's unpaired t-test and one-way ANOVA were used to compare the differences between two groups or multiple groups. Tukey's post hoc test was performed after one-way ANOVA. GraphPad Prism 8 software (GraphPad Software, Inc.) for analyzes. Data are presented as the means ± standard deviation from three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

In the present study, the effects of isoliensinine on the proliferation of human cervical cancer cell lines C33A, Caski, HeLa and SiHa cells were assessed. Isoliensinine dose-dependently inhibited the proliferation of cervical cancer cells (Fig. 1A). Following treatment with 25 µM isoliensinine, the cell viability of C33A, Caski, HeLa and SiHa cells were decreased to 12.49, 14.91, 26.16 and 39.66 at 24 h, respectively, which decreased further to 3.2, 1.78, 3.97 and 13.13 at 48 h, respectively (Fig. 1A). In addition, the IC₅₀ value was calculated to be 13.45 and 11.04 µM for HeLa at 24 and 48 h, respectively. The IC₅₀ value was calculated to be 10.27 and 7.26 µM for Caski at 24 and 48 h, respectively. By contrast, the IC₅₀ value was calculated to be 16.74 and 13.16 µM for SiHa at 24 and 48 h, respectively. The IC₅₀ value for C33A cells was calculated to be 9.53 and 7.88 µM at 24 and 48 h, respectively.

Furthermore, after isoliensinine (10 µM) treatment for 24 and 48 h, the cell viability of HeLa and SiHa cells was markedly higher compared with that of C33A cells, whilst the viability of Caski cells was comparable with that of C33A cells (Fig. 1A). Results of colony formation assay of cervical cancer cell lines C33A and HeLa revealed that isoliensinine could significantly inhibit the proliferation of cervical cancer cells (Fig. 1B). In addition, in the colony formation assay, small doses of isoliensinine (2–8 µM) was sufficient to significantly inhibit the proliferation of cervical cancer cells, suggesting that the specific mechanism may involve changes in pharmacokinetics.

To examine if the inhibitory effects of isoliensinine on proliferation was due to cell cycle arrest, the cell cycle distribution of the four cervical cancer cell lines was examined by flow cytometry. Isoliensinine treatment significantly increased G₀/G₁ cell cycle arrest in the four cervical cancer cell lines in a dose-dependent manner (Figs. 2A and B, and S1A and B). After isoliensinine treatment for 24 h, the number of cells in G₁ phase increased while those in the S and G₂ phases decreased dose-dependently (Fig. 2C). Furthermore, after 40 µM isoliensinine treatment, the percentages of C33A, Caski, HeLa and SiHa cells in G₁ phase were increased by 19.67, 14.33, 25.33 and 15.67% (Fig. 2C), respectively. By contrast, those in S phase were decreased by 7.33, 3, 10 and 5% in C33A, Caski, HeLa and SiHa cells, respectively (Fig. 2C). Those in G₂ phase were also decreased by 12.33, 11.33, 15.66 and 10.66% in C33A, Caski, HeLa and SiHa cells, respectively (Fig. 2C).

To elucidate the mechanism by which isoliensinine regulates cell cycle progression in cervical cancer cells, RT-PCR and western blotting were performed to measure the expression of cell cycle regulators (Figs. 3A and B, and S2). Isoliensinine upregulated p21 whilst downregulating CDK2 mRNA expression at the transcriptional level (Fig. 3A). Specifically, after 40 µM isoliensinine treatment, the mRNA expression of p21 were increased by 20.6-, 4.99-, 4.50- and 2.19-folds in C33A, CaSki, HeLa and SiHa cells, respectively (Fig. 3A). However, CDK2 mRNA expression was decreased by 0.48-, 0.5-, 0.41- and 0.48-folds in C33A, CaSki, HeLa and SiHa cells, respectively (Fig. 3A). Cyclin E mRNA were decreased by 0.05-, 0.07- and 0.14-folds in C33A, HeLa and SiHa cells, respectively (Fig. 3A). Changes in CDK2 and cyclin E expression were not prominent in Caski and HeLa cells on the transcriptional level (Fig. 3A). However, isoliensinine markedly reduced CDK2 and cyclin E on the protein level in the four cervical cancer cells in a dose-dependent manner (Fig. 3B).

Results from apoptosis assay revealed the dose- and time-dependent induction of apoptosis by isoliensinine in the four cervical cancer cell lines (Figs. 4A-D and S3-5). After isoliensinine (40 µM) treatment for 48 h, the percentages of apoptosis for C33A, Caski, HeLa and SiHa cells were increased by 46.60, 70.35, 23.10 and 53.63%, respectively (Fig. 4B). After isoliensinine (20 µM) treatment for 72 h, the percentages of apoptosis for these cells were increased by 66.87, 59.61, 29.78 and 50.55% in C33A, Caski, HeLa and SiHa cells, respectively (Fig. 4D).

Significant changes were not observed in the expresion of mRNA in the Bcl-2 family, namely Bcl-2, Bid, Bad and Bax, according to results from RT-PCR assay (Fig. S6A). Mcl-1 was previously reported to be highly expressed in cervical cancer tissue compared with that in normal tissue and was closely associated with the apoptosis of cervical cancer HeLa cells (28,29). Mcl-1 is a major member of the Bcl-2 family that can inhibit cell apoptosis (29). Western blotting results demonstrated that isoliensinine downregulated Mcl-1 expression and activated caspase-9 in a dose-dependent manner in SiHa and HeLa cells (Fig. 4E).

Isoliensinine was found to inhibit AKT phosphorylation and reduce GSK3α expression in the four cervical cancer cell lines in a dose- and time-dependent manner (Fig. 5A and B). However, isoliensinine downregulated AKT (S473) phosphorylation without affecting total AKT expression (Fig. 5A). Additionally, the expression of the negative regulatory factor of AKT PTEN was also mesured by RT-PCR and western blotting. Isoliensinine did not appear to induce marked changes in PTEN expression. (Figs. S2 and S6B).

To clarify the relationship between isoliensinine and AKT, the 3D structure of isoliensinine combined with the AKT1/2 proteins was analyzed using the AutoDock assay software (Fig. 6). The docking analysis showed that the docking site of isoliensinine was similar to that of AKTi-1/2. In total, the following 14 amino acids interacted with isoliensinine in AKT1: Tyr272, Asp274, Cys296, Gln79, Val270, Lys297, Leu295, Val271, Tyr18, Glu17, Gly16, Ile19, Glu85 and Thr82 (Fig. 6C). By contrast, 11 amino acids interacted with isoliensinine in AKT2: Ala173, Thr313, Glu193, Val147, Leu296, Asp275, Lys277, His355, Tyr217, Ser476 and Lys191 (Fig. 6D). Furthermore, the binding energies of isoliensinine with AKT1 and AKT2 were −7.46 and −2.31 kcal/mol, respectively. AKTi-1/2 presented prominent interactions with 13 amino acid residues in AKT1 at an affinity of −11.92 kcal/mol: Leu264, Tyr263, Ser205, Tpr80, Leu210, Thr211, Asp292, Thr82, Gly294, Ile84, Cys296, Tyr272 and Asp274 (Fig. 6E). AKTi-1/2 docked with 12 residues in AKT2 at an affinity of −5.98 kcal/mol: Ala173, Glu171, His355, Glu315, Pro314, Thr313, Leu296, Asp293, His196, Glu193, Thr197 and Lys181 (Fig. 6F).

To investigate whether the effects of isoliensinine on cell cycle arrest and apoptosis were similar to those of the AKT inhibitor AKTi-1/2, a series of experiments were conducted using AKTi-1/2 as a control in the four cervical cancer cell lines. According to previous studies and the IC₅₀ data, 5–10 µM AKTi-1/2 was selected (30,31). On the basis of ensuring that the level of apoptosis was not too high, 7.5 µM was selected. AKTi-1/2 was found to enhance the function of isoliensinine in inducing cell cycle arrest (Figs. 7A and B, and S7). After the combined treatment for 24 h, the percentages of C33A, Caski and HeLa cells in G₀/G₁ phase were increased by 13.16, 8 and 21.96%, respectively. Those in S phase were decreased by 4.1, 2.9 and 4.04%, respectively (Fig. 7B). In addition, those in the G₂ phase were decreased by 8.87, 5.23 and 17.73%, respectively (Fig. 7B). AKTi-1/2 also enhanced the ability of isoliensinine to induce apoptosis in cervical cancer cells (Fig. 8A-D). After the combined treatment for 48 h, the percentages of apoptotic C33A, Caski and HeLa cells were increased by 48.3, 77.46 and 10.06%, respectively (Fig. 8D).

To investigate if cell cycle arrest and apoptosis induced by isoliensinine and AKTi-1/2 co-treatment were related to the AKT/GSK3α pathway, the expression of AKT, phosphorylation of AKT (S473), GSK3α and p21 were measured. After treatment with isoliensinine and/or AKTi-1/2, the expression of GSK3α was decreased whilst the expression of p21 was increased in cervical cancer cells (Fig. 8E). Taken together, these data suggest that AKTi-1/2 potentiated the function of isoliensinine to induce cervical cancer cell cycle arrest and apoptosis through the AKT/GSK3α pathway in cervical cancer cells, which appeared to be an ‘additive’ effect.