Human cervical cancer cell lines SiHa (HPV 16-positive cells), HeLa (HPV 18-positive cells) and C33a (HPV negative) were provided by the Chinese Academy of Sciences (Shanghai, China). Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific, Inc. Dimethyl sulfoxide (DMSO, analytical grade) was obtained from Beijing Chemical Reagent Factory. Tan IIA was obtained from PhytoMyco, Inc. The mouse anti-human Bcl-2 antibody (cat. no. sc-23960), mouse anti-human Bax antibody (cat. no. sc-20067), rabbit ant-human cleaved caspase-3 antibody (cat. no. sc-7148), mouse anti-human cleaved caspase-9 antibody (cat. no. sc-17784), mouse anti-human cytochrome c antibody (cyto c; cat. no. sc-13561) were obtained from Santa Cruz Biotechnology, Inc. Rabbit ant-human p-AKT antibody (ID product code ab18206), rabbit ant-human AKT antibody (ID product code ab8805), rabbit ant-human p-mTOR antibody (ID product code ab84400), rabbit ant-human mTOR antibody (ID product code ab2732) were obtained from Abcam, Inc. Rabbit ant-human GLUT1 antibody (cat. no. PA5-16793), rabbit ant-human PKM2 antibody (cat. no. PA5-23034), rabbit ant-human HK2 antibodies (cat. no. PA5-29326) were purchased from Invitrogen; Thermo Fsiher Scientific, Inc.

SiHa, HeLa and C33a cells were maintained in DMEM supplemented with 10% FBS, 100 units/ml of penicillin and 100 units/ml of streptomycin at 37°C in a humidified atmosphere of 5% carbon dioxide (CO₂) and 95% air. Cells were sub-cultured every three days with 0.25% trypsin.

The cytotoxicity of Tan IIA on tumor cells was detected by MTT assay (27). SiHa, HeLa and C33a cells were seeded into a 96-well assay plate with a concentration 1.0×10⁵ cells/ml (200 µl/well). After 24 h of incubation, the cells were treated with Tan IIA with doses of 0–8 mg/l in the growth medium. Each concentration was assessed five times. The cells were cultured for another 24, 48 and 72 h. Subsequently, 20 µl of MTT (5 mg/ml) was added to each well and incubated for another 4 h. Then, supernatant was discarded and 150 µl DMSO was loaded to each well. The optical density (OD) was measured at 490 nm using a Bio-assay reader (Bio-Rad 550; Bio-Rad Laboratories, Inc.).

The total RNA form Tan IIA-treated and untreated SiHa cells were extracted to detect the expression of HPV16-E6/E7 gene. Total cellular RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) as per the manufacturer's protocol. A high-capacity cDNA reverse-transcription kit was used to prepare cDNA (Thermo Fisher Scientific, Inc). qRT-PCR was carried out using gene-specific oligonucleotide primers (Table I), an iScript One-Step RT-PCR kit with SYBR-Green and an ABI 7500 Fast real-time PCR system. The PCR conditions were set as 95°C for 5 min, followed by 40 cycles at 95°C for 30 sec, 60°C for 45 sec and 72°C for 30 min. All values were normalized to β-actin and then standardized to the control condition. Error bars represent the standard error of the mean (SEM), and the statistical significance was assessed using the Student's t-test. After completion of the RT-PCR, Cq values were obtained using the ABI 7500 fast v2.0.1 software. The ΔΔCq method was used to represent mRNA fold change (28).

SiHa cells were cultured on slides at a density of 5×10⁴/ml in 24-well plates. After Tan IIA treatment, the medium was removed and the cells were washed with PBS twice. Then, the cells were fixed with 4% paraformaldehyde for 10 min at 25°C. Subsequently, the cells were stained with Hoechst 33258 for 5 min at 25°C. The apoptosis morphology was observed using a fluorescence microscope. The images were captured at magnifications of ×200.

After Tan IIA treatment, SiHa cells were harvested by centrifugation at 1,000 × g for 3 min and washed with PBS twice. After that the harvested cells pellet was resuspend in 100 µl DMEM medium. Then 100 µl Muse Annexin V and Dead Cell reagents were added and incubated at room temperature for 20 min. The percentage of apoptosis was detected and analyzed using a Muse Cell Analyzer (EMD Millipore).

The caspase-3/7 activation change was detected by Muse Cell Analyzer following the manufacturer's instructions. After Tan IIA treatment, SiHa cells were harvested by centrifugation at 850 × g and the cell pellet was resuspended in 50 µl PBS. Muse Caspase-3/7 reagent working solution (5 µl) was added to the cell suspension, and it was mixed thoroughly and incubated at 37°C for 30 min. After incubation, 150 µl of Muse™ Caspase 7-AAD working solution was loaded to each sample, and incubated at room temperature for 5 min. Then, the caspase-3/7 activation was detected by Muse Cell Analyzer.

The SiHa cells were seeded in 6-well plates at a density of 1×10⁴ cells/well. After treatment with Tan IIA for 48 h, the cell culture media was collected to detect the glucose uptake and lactate production. For assessment of lactate production, the collected culture media was diluted 1:50 by using lactate assay buffer. The amount of lactate present in the media was then estimated using the Lactate Assay kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. The content of glucose in the collected culture media was detected immediately using a Glucose Assay kit according to the manufacturer's instructions. Glucose uptake and lactate production were all normalized by cell number (29).

SiHa cells were seeded in 6-well plates (1×10⁵ cells/ml) and then incubated with Tan IIA for 48 h. Then, the cells were harvested and lysed on ice with somatic cell ATP releasing reagent. The ATP content was detected by ATP assay kits (Genmed Scientifics, Inc.) according to the manufacturer's instructions. ATP content was normalized by cell number (29).

After Tan IIA treatment, 5×10⁵ SiHa cells were harvested and lysed in RIPA buffer (Sigma-Aldrich; Merck KGaA) to extract the total protein. The extract total protein content was determined by Bio-Rad BCA protein assay kit and 50 µg protein/lane was loaded on 12% SDS-PAGE. After SDS-PAGE, the separated proteins were transferred to nitrocellulose membranes. Non-specific binding was blocked by incubating nitrocellulose membranes in 5% non-fat dry milk in TBS/0.1% Tween for 120 min. After blocking, the blots were incubated with primary antibody: Bcl-2 antibody (dilution 1:2,000), Bax (dilution 1:2,000), cleaved caspase-3 (dilution 1:2,000), cleaved caspase-9 (dilution 1:2,000), cyto c antibody (dilution 1:2,000), p-AKT (dilution 1:1,000), AKT (dilution 1:1,000), p-mTOR (dilution 1:1,000), mTOR (dilution 1:1,000), GLUT1 (dilution 1:1,000), PKM2 (dilution 1:1,000), HK2 (dilution 1:1,000) at 4°C overnight. Then, the appropriate secondary antibody (cat. nos. sc-2357 and sc-516132; Santa Cruz Biotechnology) at a 1:5,000 dilution was added and incubation followed for 1 h at room temperature. Immunoreactive protein bands were detected by chemiluminescence using enhanced chemilumunescence reagents (ECL) was obtained from Santa Cruz Biotechnology, Inc. Blots were also stained with anti-β-actin antibody (cat. no. 60008-1-Ig; Proteintech Group) as an internal control for the amounts of target proteins. The films were then subjected to densitometry analysis using a Gel Doc 2000 system (Bio-Rad Laboratories, Inc.).

All animal experiments were performed in compliance with the guidelines of the Ethics Committee for Animal Use and Care of Beihua University. This committee approved the experiments in the present study. Kunming (KM) female mice (n=25) were purchased from Jilin University (8 weeks old; 20–25 g). The animals were maintained under a dedicated room with a 12-h light/dark cycle at a constant temperature of 22°C.

First, U14 cervical cancer cells obtained from The Chinese Academy of Sciences (Shanghai, China) were harvested by trypsin digestion, washed by PBS, and re-suspended in serum-free RPMI-1640 medium. U14 cervical cancer cell suspension (0.2 ml) with a density of 5×10⁶ was injected into the right infra-axillary dermis of the mice. When the size of the tumor reached 150 mm³, the mice were randomly assigned to 5 groups with 5 mice in each group: Control, cyclophosphamide (CTX; 25 mg/kg/day, i.g.), and three doses of Tan IIA (40 mg/kg/day; 20 mg/kg/day; and 10 mg/kg/day). The tumor volume and body weight were measured every two days for 14 days. The relative tumor volume V_t/V₀ as a function of time was used to investigate the inhibitory effect. All groups were treated every two days for 20 days, and 24 h after the last administration, the mice of the four groups were sacrificed. The tumors were excised to evaluate tumor inhibition. The tumor inhibition rate was calculated by the formula: Inhibitory rate (IR) = (the tumor weight of the control group - the tumor weight of the treated group/the tumor weight of the control group) ×100%. For euthanasia, animals were anesthetized with 5% isoflurane vaporized in oxygen and thereafter euthanatized with a 20% container volume/min gas replacement rate of carbon dioxide. Euthanasia was confirmed when the heartbeat stopped and the pupils were enlarged.

The tumor tissues were excised and fixed at 25°C for one week in 4% formalin, embedded in paraffin, and cut into 4-mm sections for histological study. Hematoxylin and eosin (H&E) purchased form Sigma-Aldrich (Merck KGaA) was used to stain the tumor for histological analysis. Histopathological analysis of the tumor tissue sections was performed using a light microscope at an ×200, magnification.

Results are presented as the mean ± SEM which are derived from three or more independent experiments. All data were analyzed by one-way ANOVA using SPSS version 13 software (SPSS, Inc.). Tukey's post hoc test was used to determine the significance for all pairwise comparisons of interest. A value of P<0.05 was considered to indicate a statistically significant difference.

An MTT assay was performed to determine the cytotoxicity of Tan IIA in SiHa, HeLa, and C33a cells after 48 h of treatment. The results revealed that Tan IIA significantly inhibited cell viability in SiHa, HeLa and C33a cells in a dose-dependent manner (Fig. 1A). Tan IIA had a greater inhibitory effect on cell viability in SiHa and HeLa cells compared with the HPV-negative C33a cells. Thus, SiHa cells were used for all subsequent experiments.

The effect of Tan IIA expression on mRNA expression of HPV16-E6/E7 in SiHa cells was determined using reverse transcription-quantitative (RT-q)PCR (Fig. 1B). Treatment with Tan IIA (1.5 mg/l) resulted in a significant reduction in the mRNA expression of HPV E6 and HPV E7. Western blot analysis also confirmed the results of the RT-qPCR (Fig. 1C).

Hoechst 333258 was used to stain apoptotic nuclei. Hoechst 333258 staining revealed that the nuclei appeared as uniform blue with organized chromatin structure in the control group (Fig. 2A). Following treatment with Tan IIA at a range of concentrations (0.5, 1 and 2 mg/l) for 48 h, the SiHa cells were decreased in number and presented with smaller nuclei, strong fluorescent spots, pyknotic nuclei and extensive blebbing. Condensed chromatin and apoptotic bodies were observed under a fluorescence microscope in the treated cells suggesting that the Tan IIA treatment had induced apoptosis in the SiHa cells. Similar results were obtained from staining with Muse Annexin V and Dead Cell reagents. As revealed in Fig. 2B and C the percentage of early apoptotic cells was increased as the dose of Tan IIA increased compared with the untreated group (P<0.01).

Muse caspase-3/7 assay kits were used to detect caspase-3/7 activation following treatment with Tan IIA in the SiHa cells. Stained cells were quantified using a Muse cell analyzer. The percentage of cells in early apoptosis was 4.9, 10.55, and 22.55% when treated with 0.5, 1 and 2 mg/l Tan IIA, respectively (Fig. 2D and E).

Tumor cells use glucose as a substrate for ATP synthesis and production. Glucose uptake in SiHa cells following treatment with Tan IIA for 48 h was measured. The results revealed that glucose uptake in Tan IIA-treated SiHa cells was decreased (Fig. 3A). The primary product of aerobic glycolysis is lactic acid and ATP. Thus, the ATP and lactic acid content were measured using reagent kits to investigate whether glycolysis inhibition was associated with Tan IIA-mediated SiHa cell apoptosis. The results revealed that the levels of intracellular ATP and extracellular lactic acid were decreased in SiHa cells treated with Tan IIA (0.5, 1 and 2 mg/l) compared with the control group (Fig. 3B and C).

Western blotting was used to assess the expression of apoptosis-associated proteins in Tan IIA-treated SiHa cells to determine which apoptosis pathway underlied Tan IIA activity in apoptosis of cervical cancer cells. As revealed in Fig. 4, after treatment with increasing doses of Tan IIA for 48 h, the expression levels of Bax, cleaved caspase-3 and cleaved caspase-9 were significantly increased. Bcl-2 expression signficantly decreased. Thus, there was a dose-dependent increase in the expression ratio of Bax/Bcl-2. In the Tan IIA-treated SiHa cells, cyto c was released from the mitochondria to the cytosol in a dose dependent manner.

The expression of GLUT1 was detected by western blotting. The western blot results revealed that Tan IIA decreased the expression of GLUT1, PKM2, and HK2 (Fig. 5), confirming inhibition of glycolysis in SiHa cells treated with Tan IIA. The AKT/mTOR pathway serves a major role in the activation of aerobic glycolysis in tumors. Therefore, it was determined whether the AKT/mTOR signaling pathway was involved in regulation of aerobic glycolysis in the Tan IIA-treated SiHa cells. Treatment with Tan IIA resulted in a dose-dependent decrease of the phosphorylation of Akt and mTOR (Fig. 6). HIF-1α mediates transformation of cell metabolism, whereas the PI3K/AKT pathways are involved in the regulation of HIF-1α. As revealed in Fig. 6, Tan IIA inhibited the expression of HIF-1α in SiHa cells.

U14 tumor-bearing Kunming mice were used to determine the antitumor effects of Tan IIA in vivo. When the the tumor size of mice reached 150 mm³, they were randomly divided into 5 groups. Mice were intraperitoneally administered with CTX (25 mg/kg) and Tan IIA (three doses) for 14 days. As revealed in Fig. 7A, the tumor in the control group grew rapidly, whereas the tumors in the mice treated with CTX and Tan IIA grew significantly slower. The body weight of the mice in the Tan IIA-treated group gradually increased but did not significantly differ from the control group, suggesting that Tan IIA did not demonstrate any significant toxic effect. At the end of the experiment, the mice were sacrificed, and the size of the tumors and tumor weights were measured. The results revealed that the tumor weight and volume in the CTX- and Tan IIA-treated groups decreased compared with the control group, in which the high dose of Tan IIA resulted in mice with a tumor weight similar to that in the CTX-treated group. The diameter of the tumor in each group is revealed in Table SI. The maximum tumor volume in each group is revealed in Table SII. The tumor inhibition rate in the CTX-treated group was 65.1%, whereas the rate in mice treated with a high dose (40 mg/kg/day) of Tan IIA was 72.7% (Fig. 7C and D and Table SIII). Pathological analysis of the tumor tissues revealed that tumors in the U14 model group exhibited invasive growth and rapid diffusion with high degrees of tumor cell proliferation. CTX-treated tumor cells exhibited unclear structures with regional necrosis, clear cell edema and a large number of disintegrated granular cells. Following treatment with Tan IIA, the tumor cells demonstrated regional necrosis, with shrunken cell volumes, nuclear condensation and clear interstitial edema occasionally infiltrated with inflammatory cells.