Human skin cancer cells A431 and SCL-1 were provided by the Dermatology Laboratory of Nanjing Medical University First Affiliated Hospital (Nanjing, China). RPMI-1640 medium was purchased from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). Fetal bovine serum, trypsin, penicillin and streptomycin were purchased from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA. Crocin and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany. Annexin V-FITC apoptosis detection kit was purchased from Bender MedSystems (Thermo Fisher Scientific, Inc.). Bid, procaspase-3, Jak2, Stat3 and Bcl-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Polyclonal goat anti-rabbit IgG-HRP secondary antibody (cat. no. sc-2004; dilution, 1:500) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Under sterile condition, 20 mg of crocin and 12.5 mg of EDTA was dissolved into 4 ml of 3-fold distilled water for stock solution with a concentration of 50 mmol/l and stored at 4°C.

A431 and SCL-2 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 U/l penicillin and 100 µg/ml streptomycin in incubator with 5% CO₂ at 37°C. The cells were subcultured routinely with trypin digestion containing 0.02% EDTA.

A431 and SCL-1 cells in the logarithmic growth phase were inoculated into cell culture plates according to the appropriate cell numbers and cultured overnight. The following day, the cells were treated with different concentrations (0, 0.4 and 0.8 mM) of crocin to detect the cell phenotypes. Alternatively, according to the instructions of Lipofectamine 2000, the cells were transfected with Jak2 overexpression plasmid (pcDNA-Jak2) for 8 h, and then replaced with complete medium for further culture. The related phenotypes were detected after the transfection process.

Cell viability was determined by MTT assay. A431 and SCL-1 cells in logarithmic growth phase were harvested for cell counting. The cell density was adjusted for a concentration of 2×10⁴cells/ml by Dulbecco's modified Eagle's medium (DMEM), then seeded into 96-well plates and incubated at 37°C in a 5% CO₂ saturated humidity incubator. After overnight adherence, the supernatant was aspirated and different concentrations of crocin (concentration 0, 0.2, 0.4, 0.8 and 1.0 mmol/l) were added to corresponding treatments for another 24 h incubation. On the other hand, the same concentration (0.8 mmol/l) of crocin was added and cultured for 0, 6, 12, 24, 48 and 72 h, respectively. A total of 20 µl MTT (5 g/l) solution was added to each well for 4 h and centrifuged at 1,750 × g for 10 min at 4°C. The reaction was terminated by discarding the supernatant and adding 150 µl dimethylsulphoxide (DMSO) per-well. The sample was placed on a shaker at shaking speed for 10 min. The OD value was measured using spectrophotometer (Hitachi, Ltd., Tokyo, Japan) at 570 nm wavelength. Each treatment was set up in triplicates. Inhibition rate was calculated as follows: Inhibition rate (%) = (control group A - experimental group A)/control group A ×100%.

Cells in logarithmic growth phase were converted into cell suspension status by using conventional digestion and passage method. The cells were dissociated thoroughly and repeatedly to make the cell percentage of each single cell above 95%. A total of 200 cells were taken and seeded in 6-well plates with 2 ml medium and shaken gently on a shaker. Afterwards the plate was placed and incubated at 37°C in a 5% CO₂ saturated humidity incubator for another 2 weeks. The formation of clones was monitored under a microscope (BX-42; Olympus Corporation, Tokyo, Japan). The culture medium was removed when clones were grown to the appropriate number and size. Cells were fixed by adding 4% polyoxymethylene for 10 min, and then rinsed with phosphate-buffered saline (PBS) twice. After stained with hematoxylin for 10 min, the cells were rinsed by PBS and air dried. After that the cells were photographed and counted under the microscope (BX-42; Olympus Corporation).

EDU kit was used for the experiment. Cells in logarithmic growth phase were collected for routine digestion, centrifugation, resuspension and counting. Harvested cells were seeded into 96-well plates at a density of 4×10³ cells/well. After the cells grew adherently and in proper concentration, EDU staining procedure was performed following the kit instructions. After staining the cells were photographed and counted under the fluorescence microscope (IX70; Olympus Corporation). Samples that appeared in >3 random sights were selected and calculated utilizing IPWIN60 software for the number of cells in S phase out of every 200 cells. The ratio was calculated by dividing the number of cells in S phase by 200, and then by further statistical analysis.

The human skin cancer cells were collected and adjusted to a concentration of 1×10⁵/ml, and then seeded on 6-well plates with 2 ml per well. After the cells were incubated for 24 h, supernatant was removed and serum-free medium containing 0, 0.4, 0.8 mmol/l of crocin was added. The 6-well plates were placed into the incubator again for another 24 h culture for following tests. For apoptosis assay, the cells were washed twice with PBS, then collected at a density of 5×10⁵. After centrifugation at 1,500 × g for 10 min at 20°C, 500 µl of binding buffer was added to resuspend cells. A total of 5 µl of Annexin V-FITC and 5 µl of pro-pidium iodide (PI) was added and mixed. The reaction was protected from light at room temperature for 10 min. Finally, apoptosis index was evaluated utilizing the flow cytometer. To detect the cell cycle phrase distribution, the cells were washed once with PBS and then collected and adjusted to a concentration of 1×10⁶/ml after centrifugation. The sample was stabilized with 70% ethanol and preserved at 4°C. The fixative was then washed with PBS before staining. A total of 100 µl RNase A was added and heated up to 37°C in water bath for approximately 30 min. Then the sample was treated with 400 µl PI staining mix and incubated in the dark at 4°C. After 30 min, the red fluorescence at 488 nm wavelength was evaluated and recorded utilizing the flow cytometer.

According to the amount of cells, appropriate amount of RIPA cell lysis solution was added. RIPA lysis and extraction buffer (cat. no. 89900; Thermo Fisher Scientific, Waltham, MA, USA) was used for the western blot analysis. Cell lysate was collected, sonicated on ice for 1 min, and centrifuged. After centrifugation at 10,000 × g for 10 min at 4°C, the supernatant was collected to determine the protein concentration by bicincho-ninic acid (BCA) method. A total of 30 µg protein sample was loaded on 10% sodium dodecyl sulphate (SDS) gel for each well. After the electrophoresis, the gel was transferred to the polyvinylidene fluoride (PVDF) membrane and blocked using 5% fat-free milk for 1 h. After that, corresponding primary antibodies was added to each corresponding sample and incubated at 4°C overnight. Then the corresponding secondary antibody were added for 1 h incubation at room temperature. Primary mouse monoclonal B-cell lymphoma-2 (Bcl-2) antibody (cat. no. ab59348; dilution, 1:500); rabbit polyclonal Caspase-3 antibody (cat. no. ab13847; dilution, 1:500); rabbit monoclonal Bid antibody (cat. no. ab32060; dilution, 1:500); rabbit monoclonal JAK2 antibody (cat. no. ab108596; dilution, 1:500); mouse monoclonal STAT3 antibody (cat. no. ab119352; dilution, 1:500); rabbit polyclonal GAPDH antibody (cat. no. ab37168; dilution, 1:500) and secondary goat anti-rabbit (HRP) IgG antibody (cat. no. ab6721; dilution, 1:2,000) were all purchased from Abcam (Cambridge, MA, USA). Lastly enhanced chemiluminescent method was used to expose the protein band. The experiment was repeated three times independently.

The measurement data were expressed as mean ± standard deviation, utilizing SPSS 11.0 (SPSS Inc., Chicago, IL, USA) software for statistical analysis. t-test was used to compare two groups. P<0.05 was considered to indicate a statistically significant difference.

Crocin significantly inhibited the proliferation of A431 and SCL-1 cells and in a dose-dependent manner. The maximum inhibitory effect appeared at 1 mM (Fig. 1A), and the effect increased with the training time (Fig. 1B). Crocin was also found capable of constraining the clonogenic capacity of human skin cancer cells. Also, the group using the concentration of 0.8 mmol/l crocin had enhanced inhibitory effects compared to 0.4 mmol/l (Fig. 1C). Furthermore, EDU experimental results showed that crocin can inhibit the viability of human skin cancer cells in a concentration-dependent manner (Fig. 1D). In conclusion, the above results showed that crocin has potential therapeutic effect on skin cancer.

Flow cytometry analysis showed that crocin could induce apoptosis of A431 and SCL-1 cells to a certain extent, which was positively correlated with the concentration (Fig. 2A). Moreover, compared with the control group, it was found that crocin could arrest A431 and SCL-1 cells in G0/G1 phase (Fig. 2B).

A431 and SCL-1 cells were cultured individually with 0, 0.4, 0.8 mmol/l crocin. The proteins were collected after 24 h to evaluate the expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein Bid and procaspase-3. The results showed that the expression of anti-apoptotic protein was downregulated, while the expression of pro-apoptotic protein was upregulated. Also, the group using the concentration of 0.8 mmol/l crocin exerted better effects than that of 0.4 mmol/l. These results suggested that crocin induced apoptosis of skin cancer cells A431 and SCL-1 by regulating apoptosis pathway (Fig. 3).

A431 and SCL-1 cells were cultured in medium containing 0, 0.4 and 0.8 mmol/l crocin respectively for the purpose of evaluating the expression of Jak2 and Stat3 proteins using western blot analysis. The results showed that crocin can inhibit the expression of Jak2 and Stat3 protein in a dose-dependent pattern (Fig. 4A). After crocin treatment and overexpressing Jak2 at the same time, cell viability result by MTT assay showed that the cell viability of 0.8 mmol/l crocin group showed a significant decrease at 24, 48 and 72 h compared with the control group. The overexpression of Jak2 partially reversed the inhibitory effects of crocin, further suggesting that crocin could inhibit the Jak2/Stat3 pathway in human skin cancer cells to facilitate apoptosis (Fig. 4B).